Qualitative differential screening

ABSTRACT

The invention concerns a method for identifying and/or cloning nucleic acid regions representing qualitative differences associated with alternative splicing events and/or with insertions, deletions located in RNA transcribed genome regions, between two physiological situations, comprising either hybridization of RNA derived from the test situation with cDNA&#39;s derived from the reference situation and/or reciprocally, or double-strand hybridization of cDNA derived from the test situation with cDNA&#39;s derived from the reference situation; and identifying and/or cloning nucleic acids representing qualitative differences. The invention also concerns compositions or banks of nucleic acids representing qualitative differences between two physiological situations, obtainable by the above method, and their use as probe, for identifying genes or molecules of interest, or still for example in methods of pharmacogenomics, and profiling of molecules relative to their therapeutic and/or toxic effects. The invention further concerns the use of dysregulation of splicing RNA as markers for predicting molecule toxicity and/or efficacy, and as markers in pharmacogenomics.

This application is continuation-in-part of U.S. Ser. No. 09/046,920,filed Mar. 24, 1998, now U.S. Pat. No. 6,251,590.

The present invention relates to the fields of biotechnology medicine,biology and biochemistry. Applications thereof are aimed at humanhealth, animal and plant care. More particularly, the invention makes itpossible to identify nucleic acid sequences whereby both novel screeningmethods for identifying molecules of therapeutic interest and novel genetherapy tools can be developed, and it further provides information onthe toxicity and potency of molecules, as well as pharmacogenomic data.

The present invention primarily describes a set of original methods foridentifying nucleic acid sequences which rely on demonstratingqualitative differences between RNAs derived from two distinct statesbeing compared, in particular those derived from a diseased organ ortissue and healthy equivalents thereof. More specifically, these methodsare intended to specifically clone alternative exons and introns whichare differentially spliced with respect to a pathological condition anda healthy state or with respect to two physiological conditions onewishes to compare. These qualitative differences in RNAs can also be dueto genome alterations such as insertions or deletions in the regions tobe transcribed to RNA. This set of methods is identified by the acronymDATAS: Differential Analysis of Transcripts with Alterative Splicing.

The characterization of gene expression alterations which underly or arelinked to a given disorder raises substantial hope regarding thediscovery of novel therapeutic targets and of original diagnostic tools.However, the identification of a genomic or complementary DNA sequence,whether through positional cloning or quantitative differentialscreening techniques, yields little, if any, information on thefunction, and even less on the functional domains, involved in theregulation defects related to the disease under study. The presentinvention describes a set of original methods aimed at identifyingdifferences in RNA splicing occurring between two distinctpathophysiological conditions. Identifying such differences providesinformation on qualitative but not on quantitative differences as hasbeen the case for techniques described so far. The techniques disclosedin the present invention are hence all encompassed under the term of“qualitative differential screening”, or DATAS. The methods of theinvention may be used to identify novel targets or therapeutic products,to devise genetic research and/or diagnostic tools, to construct nucleicacid libraries, and to develop methods for determining the toxicologicalprofile or potency of a compound for example.

A first object of the invention is based more particularly on a methodfor identifying and/or cloning nucleic acid regions which correspond toqualitative genetic differences occurring between two biologicalsamples, comprising hybridizing a population of double stranded cDNAs orRNAs derived from a first biological sample, with a population of cDNAsderived from a second biological sample (FIG. 1A).

As indicated hereinabove, the qualitative genetic differences may be dueto alterations of RNA splicing or to deletions and/or insertions in theregions of the genome which are transcribed to RNA.

In a first embodiment, the hybridization is carried out between RNAsderived from a first biological sample and cDNAs (single stranded ordouble stranded) derived from a second biological sample.

In another embodiment, the hybridization is carried out between doublestranded cDNAs derived from a first biological sample, and cDNAs (doublestranded or, preferably, single stranded) derived from a secondbiological sample.

A more specific object of the invention is to provide a method foridentifying differentially spliced nucleic acid regions occurringbetween two physiological conditions, comprising hybridizing apopulation of RNAs or double stranded cDNAs derived from a testcondition with a population of cDNAs originating from a referencecondition and identifying nucleic acids which correspond to differentialsplicing events.

Another object of the invention is to provide a method for cloningdifferentially spliced nucleic acids occurring between two physiologicalconditions, comprising hybridizing a population of RNAs or doublestranded cDNAs derived from the test condition with a population ofcDNAs originating from the reference condition and cloning nucleic acidswhich correspond to differential splicing events.

In a particular embodiment, the method of nucleic acid identificationand/or cloning according to the invention comprises running twohybridizations in parallel consisting of:

(a) hybridizing RNAs derived from the first sample (test condition) withcDNAs derived from the second sample (reference condition);

(b) hybridizing RNAs derived from the second sample (referencecondition) with cDNAs derived from the first sample (test condition);and

(c) identifying and/or cloning, from the hybrids formed in steps (a) and(b), those nucleic acids corresponding to qualitative geneticdifferences.

The present invention is equally directed to the preparation of nucleicacid libraries, to the nucleic acids and libraries thus prepared, aswell as to uses of such materials in all fields ofbiology/biotechnology, as illustrated hereinafter.

In this respect, the invention is equally directed to a method forpreparing profiled nucleic acid compositions or libraries,representative of qualitative differences occurring between twobiological samples, comprising hybridizing RNAs derived from a firstbiological sample with cDNAs originating from a second biologicalsample.

The invention further concerns a method for profiling a cDNAcomposition, comprising hybridizing this composition with RNAs, or viceversa.

As indicated hereinabove, the present invention relates in particular tomethods for identifying and cloning nucleic acids representative of aphysiological state. In addition, the nucleic acids identified and/orcloned represent the qualitative characteristics of a physiologicalstate in that these nucleic acids are generally involved to a greatextent in the physiological state being observed. Thus, the qualitativemethods of the invention afford direct exploration of genetic elementsor protein products thereof, playing a functional role in thedevelopment of a pathophysiological state.

The methods of the invention are partly based on an original stepconsisting of cross hybridization between RNAs and cDNAs belonging todistinct physiological states. This or these cross hybridizationprocedures advantageously allow one to demonstrate, in the hybridsformed, unpaired regions, i.e. regions present in RNAs in a givenphysiological condition and not in RNAs from another physiologicalcondition. Such regions essentially correspond to alternative forms ofsplicing typical of a physiological state, but may also be a reflectionof genetic alterations such as insertions or deletions, and thus formgenetic elements particularly useful in the fields of therapeutics anddiagnostics as set forth below. The invention therefore consists notablyin keeping the complexes formed after cross hybridization(s), so as todeduce therefrom the regions corresponding to qualitative differences.This methodology can be distinguished from quantitative subtractiontechniques known to those skilled in the art (Sargent and Dawid (1983),Science, 222: 135-139; Davis et al. (1984), PNAS, 81: 2194-2198; Duguidand Dinauer (1990), Nucl. Acid Res., 18: 2789-2792; Diatchenko et al.(1996), PNAS, 93: 6025-6030), which discard the hybrids formed afterhybridization(s) so as to conserve only the non-hybridized nucleicacids.

The invention therefore first deals with a method for identifyingnucleic acids of interest comprising hybridizing the RNAs of a testsample with the cDNAs of a reference sample. This hybridizationprocedure makes it possible to identify, in the complexes formed,qualitative genetic differences between the conditions under study, andthus to identify and/or clone for example the splicings which arecharacteristic of the test condition.

According to a first variant of the invention, the method thereforeallows one to generate a nucleic acid population characteristic ofsplicing events that occur in the physiological test condition ascompared to the reference condition (FIGS. 1A, 1B). As indicatedhereinafter, this population can be used for the cloning andcharacterization of nucleic acids, their use in diagnostics, screening,therapeutics and antibody production or synthesis of whole proteins orprotein fragments. This population can also be used to generatelibraries that may be used in different fields of application as shownhereinafter and to generate labeled probes (FIG. 1D).

According to another variant of the invention, the method comprises afirst hybridization as described hereinbefore and a secondhybridization, conducted in parallel, between RNAs derived from thereference condition and cDNAs derived from the test condition. Thisvariant is particularly advantageous since it allows one to generate twonucleic acid populations, one representing the qualitativecharacteristics of the test condition with respect to the referencecondition, and the other representing the qualitative characteristics ofthe reference condition in relation to the test condition (FIG. 1C).These two populations can also be utilized as nucleic acid sources, oras libraries which serve as genetic fingerprints of a particularphysiological condition, as will be more fully described in thefollowing (FIG. 1D).

The present invention may be applied to all types of biological samples.In particular, the biological sample can be any cell, organ, tissue,sample, biopsy material, etc. containing nucleic acids. In the case ofan organ, tissue or biopsy material, the samples can be cultured so asto facilitate access to the constituent cells. The samples may bederived from mammals (especially human beings), plants, bacteria andlower eukaryotes (yeasts, fungal cells, etc.). Relevant materials areexemplified in particular by a tumor biopsy, neurodegenerative plaque orcerebral zone biopsy displaying neurodegenerative signs, a skin sample,a blood sample obtained by collecting blood, a colorectal biopsy, biopsymaterial derived from bronchoalveolar lavage, etc. Examples of cellsinclude notably muscle cells, hepatic cells, fibroblasts, nerve cells,epidermal and dermal cells, blood cells such as B and T lymphocytes,mast cells, monocytes, granulocytes and macrophages.

As indicated hereinabove, the qualitative differential screeningaccording to the present invention allows the identification of nucleicacids characteristic of a given physiological condition (condition B) inrelation to a reference physiological condition (condition A), that areto be cloned or used for other applications. By way of illustration, thephysiological conditions A and B being investigated may be chosen amongthe following:

CONDITION A CONDITION B Healthy subject-derived sample Pathologicalsample Healthy subject-derived sample Apoptotic sample Healthysubject-derived sample Sample obtained after viral infection X-sensitivesample X-resistant sample Untreated sample Treated sample (for exampleby a toxic compound) Undifferentiated sample Sample that has undergonecellular or tissue differentiationRNA Populations

The present invention can be carried out by using total RNAs ormessenger RNAs. These RNAs can be prepared by any conventional molecularbiology methods, familiar to those skilled in the art. Such methodsgenerally comprise cell, tissue or sample lysis and RNA recovery bymeans of extraction procedures. This can be done in particular bytreatment with chaotropic agents such as guanidium thiocyanate (whichdisrupts the cells without affecting RNA) followed by RNA extractionwith solvents (phenol, chloroform for instance). Such methods are wellknown in the art (see Maniatis et al., Chomczynski et al., (1987), Anal.Biochem., 162: 156). These methods may be readily implemented by usingcommercially available kits such as for example the US73750 kit(Amersham) or the Rneasy kit (Quiagen) for total RNAs. It is notnecessary that the RNA be in a fully pure state, and in particular,traces of genomic DNA or other cellular components (protein, etc.)remaining in the preparations will not interfere, in as much as they donot significantly affect RNA stability and as the modes of preparationof the different samples under comparison are the same. Optionally, itis further possible to use messenger RNA instead of total RNApreparations. These may be isolated, either directly from the biologicalsample or from total RNAs, by means of polyT sequences, according tostandard methods. In this respect, the preparation of messenger RNAs canbe carried out using commercially available kits such as for example theUS72700 kit (Amersham) or the kit involving the use of oligo-(dT) beads(Dynal). An advantageous method of RNA preparation consists inextracting cytosolic RNAs and then cytosolic polyA+ RNAs. Kits allowingthe selective preparation of cytosolic RNAs that are not contaminated bypremessenger RNAs bearing unspliced exons and introns are commerciallyavailable. This is the case in particular for the Rneasy kit marketed byQiagen (example of catalog number: 74103). RNAs can also be obtaineddirectly from libraries or other samples prepared beforehand and/oravailable from collections, stored under suitable conditions.

Generally, the RNA preparations used advantageously comprise at least0.1 μg of RNA, preferably at least 0.5 μg of RNA. Quantities can varydepending on the particular cells and methods being used, while keepingthe practice of the invention unchanged. In order to obtain sufficientquantities of RNA (preferably at least 0.1 μg), it is generallyrecommended to use a biological sample including at least 10⁵ cells. Inthis respect, a typical biopsy specimen generally comprises from 10⁵ to10⁸ cells, and a cell culture on a typical petri dish (6 to 10 cm indiameter) contains about 10⁶ cells, so that sufficient quantities of RNAcan be readily obtained.

The RNA preparations may be used extemporaneously or stored, preferablyin a cold place, as a solution or in the frozen state, for later use.

cDNA Populations

The cDNA used within the scope of the present invention may be obtainedby reverse transcription according to conventional molecular biologytechniques. Reference is made in particular to Maniatis et al. Reversetranscription is generally carried out using an enzyme, reversetranscriptase, and a primer.

In this respect, many reverse transcriptases have been described in theliterature and are commercially available (1483188 kit, Boehringer).Examples of the most commonly employed reverse transcriptases includethose derived from avian virus AMV (Avian Myeloblastosis Virus) and frommurine leukemia virus MMLV (Moloney Murine Leukemia Virus). It is alsoworth mentioning certain thermostable DNA polymerases having reversetranscriptase activity such as those isolated from Thermus flavus andThermus thermophilus HB-8 (commercially available; Promega catalognumbers M1941 and M2101). According to an advantageous variant, thepresent invention is practiced using AMV reverse transcriptase sincethis enzyme, active at 42° C. (in contrast to that of MMLV which isactive at 37° C.), destabilizes certain RNA secondary structures thatmight stop elongation, and therefore allows reverse transcription of RNAof greater length, and provides cDNA preparations in high yields thatare much more faithful copies of RNA.

According to a further advantageous variant of the invention, a reversetranscriptase devoid of RNaseH activity is employed. The use of thistype of enzyme has several advantages, particularly that of increasingthe yield of cDNA synthesis and avoiding any degradation of RNAs, whichwill then be engaged in heteroduplex formation with the newlysynthesized cDNAs, thereby optionally making it possible to omit thephenol extraction of the latter. Reverse transcriptases devoid of RNaseHactivity may be prepared from any reverse transcriptase by deletion(s)and/or mutagenesis. In addition, such enzymes are also commerciallyavailable (for example Life Technologies, catalog number 18053-017).

The operating conditions that apply to reverse transcriptases(concentration and temperature) are well known to those skilled in theart. In particular, 10 to 30 units of enzyme are generally used in asingle reaction, in the presence of an optimal Mg²⁺ concentration of 10mM.

The primer(s) used for reverse transcription may be of various types. Itmight be, in particular, a random oligonucleotide comprising preferablyfrom 4 to 10 nucleotides, advantageously a hexanucleotide. Use of thistype of random primer has been described in the literature and allowsrandom initiation of reverse transcription at different sites within theRNA molecules. This technique is especially employed for reversetranscribing total RNA (i.e. comprising mRNA, tRNA and rRNA inparticular). Where it is desired to carry out reverse transcription ofmRNA only, it is advantageous to use an oligo-dT oligonucleotide asprimer, which allows initiation of reverse transcription starting frompolyA tails specific to messenger RNAs. The oligo-dT oligonucleotide maycomprise from 4 to 20-mers, advantageously about 15-mers. Use of such aprimer represents a preferred embodiment of the invention. In addition,it might be advantageous to use a labeled primer for reversetranscription. As a matter of fact, this allows recognition and/orselection and/or subsequent sorting of RNA from cDNA. This may alsoallow one to isolate RNA/DNA heteroduplexes the formation of whichrepresents a crucial step in the practice of the invention. Labeling ofthe primer may be done by any ligand-receptor based system, i.e.providing affinity mediated separation of molecules bearing the primer.It may consist for instance of biotin labeling, which can be captured onany support (bead, column, plates, etc.) previously coated withstreptavidin. Any other labeling system allowing separation withoutaffecting the properties of the primer may be likewise utilized.

In typical operating conditions, this reverse transcription generatessingle stranded complementary DNA (cDNA). This represents a firstadvantageous embodiment of the present invention.

In a second variant of practicing the invention, reverse transcriptionis accomplished such that double stranded cDNAs are prepared. Thisresult is achieved by generating, following transcription of the firstcDNA strand, the second strand using conventional molecular biologytechniques involving enzymes capable of modifying DNA such as phage T4DNA ligase, DNA polymerase I and phage T4 DNA polymerase.

The cDNA preparations may be used extemporaneously or stored, preferablyin a cold place, as a solution or in the frozen state, for later use.

Hybridizations

As set forth hereinabove, the methods according to the invention arepartly based on an original cross hybridization step between RNAs andcDNAs derived from biological samples in distinct physiologicalconditions or from different origins. In a preferred embodiment,hybridization according to the invention is advantageously performed inthe liquid phase. Furthermore, it may be carried out in any appropriatedevice, such as for example tubes (Eppendorff tubes, for instance),plates or any other suitable support that is commonly used in molecularbiology. Hybridization is advantageously carried out in volumes rangingfrom 10 to 1000 μl, for example from 10 to 500 μl. It should beunderstood that the particular device as well as the volumes used can beeasily adapted by those skilled in the art. The amounts of nucleic acidsused for hybridization are equally well known in the art. In general, itis sufficient to use a few micrograms of nucleic acids, for example inthe range of 0.1 to 100 μg.

An important factor to be considered when performing hybridization isthe respective quantities of nucleic acids used. Thus, it is possible touse nucleic acids in a cDNA/RNA ratio ranging from 50 to 0.02approximately, preferably from 40 to 0.1. In a more particularlyadvantageous manner, the cDNA/RNA ratio is preferably close to orgreater than 1. Indeed, in such experiments, RNA forms the testercompound and cDNA forms the driver. Accordingly, in order to improve thespecificity of the method, it is preferred to choose operatingconditions where the driver is in excess relative to the tester. Infact, in such conditions, the cooperativity effect between nucleic acidsoccurs and mismatches are strongly disfavored. As a result, the onlymismatches that are observed are generally due to the presence ofregions in the tester RNA which are absent from the driver cDNA andwhich can therefore be considered as specific. In order to enhance thespecificity of the method, hybridization is therefore advantageouslyperformed using a cDNA/RNA ratio comprised between about 1 and about 10.It is understood that this ratio can be adapted by those skilled in theart depending on the operating conditions (nucleic acid quantitiesavailable, physiological conditions, required results, etc.). The otherhybridization parameters (time, temperature, ionic strength) are alsoadaptable by those skilled in the art. Generally speaking, afterdenaturation of the tester and driver (by heating for instance),hybridization is accomplished for about 2 to 24 hours, at a temperatureof approximately 37° C. (and by optionally performing temperature shiftsas set forth below), and under standard ionic strength conditions(ranging from 0.1 M to 5 M NaCl for instance). It is known that ionicstrength is one of the factors that defines hybridization stringency,notably in the case of hybridization on a solid support.

According to a specific embodiment of the invention, hybridization iscarried out in phenol emulsion, for instance according to the PERTtechnique (Phenol Emulsion DNA Reassociation Technique) described byKohne D. E. et al. (Biochemistry, (1977), 16 (24): 5329-5341).Advantageously, use is made within the scope of the present invention ofphenol emulsion hybridization under temperature cycling (temperatureshifts from about 37° C. to about 60/65° C.) instead of stirring,according to the technique of Miller and Riblet (NAR, (1995), 23: 2339).Any other liquid phase hybridization technique, notably in emulsionphase, may be used within the scope of the present invention. Thus, inanother particularly advantageous embodiment, hybridization is carriedout in a solution containing 80% formamide, at a temperature of 40° C.for instance.

Hybridization may also be carried out with one of the partners fixed toa support Advantageously, the cDNA is immobilized. This may be done bytaking advantage of cDNA labeling (see hereinabove), especially by usingbiotinylated primers. Biotin moieties are contacted with magnetic beadscoated with streptavidin molecules. cDNAs can then be held in contactwith the filter or the microtiter dish well by applying a magneticfield. Under appropriate ionic strength conditions, RNAs aresubsequently contacted with cDNAs. Unpaired RNAs are eliminated bywashing. Hybridized RNAs as well as cDNAs are recovered upon removal ofthe magnetic field.

Where the cONA is double stranded, the hybridization conditions used areessentially similar to those described hereinabove, and adaptable bythose skilled in the art. In this case, hybridization is preferablyperformed in the presence of formamide and the complexes are exposed toa range of temperatures varying for instance from 60 to 40° C.,preferably from 56° C. to 44° C., so as to promote the formation ofR-loop complexes. In addition, it is desirable to add, followinghybridization, a stabilizing agent to stabilize the triplex structuresformed, once formamide is removed from the medium, such as glyoxal forexample (Kaback et al., (1979), Nuc. Acid Res., 6: 2499-2517).

These cross hybridizations according to the invention thus generatecompositions comprising cDNA/RNA heteroduplex or heterotriplexstructures, representing the qualitative properties of eachphysiological condition being tested. As already noted, in each of thepresent compositions, nucleic acids essentially corresponding todifferential alternative splicing or to other genetic alterations,specific to each physiological condition, can be identified and/orcloned.

The invention therefore advantageously relates to a method foridentifying and/or cloning nucleic acid regions representative ofgenetic differences occurring between two physiological conditions,comprising hybridizing RNAs derived from a biological sample in a firstphysiological condition with single stranded cDNAs derived from abiological sample in a second physiological condition, and identifyingand/or cloning, from the hybrids thus formed, unpaired RNA regions.

This first variant is more specifically based upon the formation ofheteroduplex structures between RNAs and single stranded cDNAs (seeFIGS. 2-4). This variant is advantageously implemented using messengerRNAs or cDNAs produced by reverse transcription of essentially messengermRNAs, i.e. in the presence of an oligo-dT primer.

In a particular embodiment, the method for identifying and/or cloningnucleic acids according to the invention comprises:

(a) hybridizing RNAs derived from the test condition with singlestranded cDNAs derived from the reference condition;

(b) hybridizing RNAs derived from the reference condition with singlestranded cDNAs derived from the test condition; and

(c) identifying and/or cloning, from the hybrids formed in steps (a) and(b), unpaired RNA regions.

In a particular alterative mode of execution, the method of theinvention comprises the following steps:

(a) obtaining RNAs from a biological sample in a physiological conditionA (rA);

(b) obtaining RNAs from an identical biological sample in aphysiological condition B (rB);

(c) preparing cDNAs from a portion of rA RNAs provided in step (a) (cAcDNAs) and from a portion of rB RNAs provided in step B (cB cDNAs) bymeans of polyT primers,

(d) hybridizing in liquid phase a portion of rA RNAs with a portion ofcB DNAs (to generate rA/cB heteroduplexes)

(e) hybridizing in liquid phase a portion of rB RNAs with a portion ofcA DNAs (to generate rB/cA heteroduplexes),

(f) identifying and/or cloning unpaired RNA regions within the rA/cB andrB/cA heteroduplexes obtained in steps (d) and (e).

According to an alternative mode of practicing the invention, the methodof the invention comprises hybridizing RNAs derived from the testcondition with double stranded cDNAs derived from the referencecondition, and identifying and/or cloning the resulting double strandedDNA regions. This second variant is more specifically based upon theformation of heterotriplex structures between RNAs and double strandedcDNAs, derived from R-loop type structures (see FIG. 5). This variant isequally preferentially practiced by using messenger RNAs or cDNAsproduced by reverse transcription of essentially messenger RNA, i.e. inthe presence of a polyT primer. In this variant again, a particularembodiment comprises running two hybridizations in parallel, whereby twonucleic acid populations according to the invention are generated. Inthis variant, the desired regions, specific of alternative splicingevents, are not the unpaired RNA regions, but instead double strandedDNA which was not displaced by a homologous RNA sequence (see FIG. 5).

In another variant of the invention, the method to detect qualitativegenetic differences (eg., alternative splicing events) occurring betweentwo samples, comprises hybridizing double stranded cDNAs derived from afirst biological sample with cDNAs (double stranded or, preferablysingle stranded) derived from a second biological sample (FIG. 6).

A Unlike the variants described hereinabove, this variant does not makeuse of DNA/RNA heteroduplex or heterotriplex structures, but instead ofDNA/DNA homoduplexes. This variant is advantageous in that it revealsnot only alternative introns and exons but also, and within a samenucleic acid library, specific junctions formed by deletion of an exonor an intron. Furthermore, the sequences in such a library giveinformation about the flanking sequences of alternative introns andexons.

For both samples (i.e. pathophysiological conditions) under study,cytosolic polyA+ RNAs are extracted by techniques known in the art anddescribed previously. These RNAs are converted to cDNA through theaction of a reverse transcriptase with or without intrinsic RNase Hactivity, as described hereinabove. One of these single stranded cDNAsis then converted to double stranded cDNA by priming with randomhexamers and according to techniques known to those skilled in the art.For one of the conditions under study one therefore has a singlestranded cDNA (called a “driver”) and for the other condition, adouble-stranded cDNA (called a “tester”). These cDNAs are denatured byheating and then mixed such that the driver is in excess relative to thetester. This excess is chosen between 1 and 50-fold, advantageously10-fold. In a given experiment, conducted starting with twopathophysiological conditions, the choice of the condition whichgenerates the driver is arbitrary and must not affect the nature of thedata collected. As a matter of fact, as in the case of the approachesdescribed hereinabove, the strategy for identifying qualitativedifferences occurring between two mRNA populations is based on cloningthese differences present in common messengers: the strategy is based oncloning sequences present within duplexes instead of single strandscorresponding to unique sequences or sequences in excess in one of theconditions under study. The mixture of cDNAs is precipitated, then takenup in a solution containing formamide (for example, 80%). Hybridizationis carried out for 16 hours to 48 hours, advantageously for 24 hours.The hybridization products are precipitated, then subjected to theaction of a restriction endonuclease having a 4-base recognition sitefor double stranded DNA. Such a restriction enzyme will therefore cleavethe double stranded cDNA formed during the hybridization on averageevery 256 bases. This enzyme is advantageously chosen so as to generatecohesive ends. Such enzymes are exemplified by restriction enzymes suchas Sau3AI, HpaII, TaqI and MseI. The double stranded fragments digestedby these enzymes are therefore accessible to a cloning strategy makinguse of the cleaved restriction sites. Such fragments are of two types:fully hybridized fragments, the two strands of which are fullycomplementary, and partially hybridized fragments, i.e. comprising asingle stranded loop flanked by double stranded regions (FIG. 6A). Theselatter fragments, which are in the minority, contain the information ofinterest. In order to separate them from fully hybridized fragments,which are in the majority since they are derived from most of the cDNAlength, separation methods on a gel or on any other suitable matrix areused. These methods take advantage of the slower migration, duringelectrophoreis or gel filtration in particular, of DNA fragments whichcontain a single stranded DNA loop. In this manner the minorityfragments which contain the desired information can be preparativelyseparated from the majority of fragments corresponding to identical DNAregions in both populations. This variant, which makes it possible toisolate, from a same population, positive and negative fingerprintslinked to qualitative differences, can also be practiced with RNA/DNAheteroduplex structures. In this respect, an example of slower migrationof a RNA/DNA heteroduplex in which a portion of the RNA is not paired,as compared to a homologous heteroduplex in which all the sequences arepaired, is illustrated in the grb2/grb33 model described in the examples(in particular see FIG. 8, lanes 2 and 3).

Identification and/or Cloning

Starting from nucleic acid populations generated by hybridization, theregions characterizing qualitative differences (eg., differentialalternative splicing events), may be identified by any technique knownto those skilled in the art.

Identification and/or cloning starting with RNA/DNA heteroduplexes

Hence, in case of an RNA/DNA heteroduplex (first variant of thismethod), these regions essentially appear as unpaired RNA regions (RNAloops), as shown in FIG. 3. These regions may thus be identified andcloned by separating the heteroduplexes and single stranded nucleicacids (DNA, RNA) (unreacted nucleic acids in excess), selectivelydigesting the double stranded RNA (portions engaged in heteroduplexstructures) and finally separating the resulting single stranded RNAfrom the single stranded DNA.

In this respect, according to a first approach illustrated in FIG. 3,the unpaired RNA regions are identified by treatment of heteroduplexesby means of an enzyme capable of selectively digesting the RNA domainsengaged in RNA/DNA heteroduplexes. Enzymes having such activity areknown from the prior art and are commercially available. It can bementioned RNases H, such as in particular, those derived from E. coli byrecombinant techniques and commercially available (Promega catalognumber M4281; Life Technologies catalog number 18021). This firsttreatment thus generates a mixture comprising unpaired single strandedRNA regions and single stranded cDNA. The RNAs may be separated fromcDNAs by any technique known in the art, and notably on the basis oflabeling of those primers used to prepare cDNA (see above). These RNAscan be used as a source of material for identifying targets, geneproducts of interest or for any other application. These RNAs can beequally converted into cDNA, and then cloned into vectors, as describedhereinafter.

In this regard, cloning RNAs may be done in different ways. One way isto insert at each RNA end oligonucleotides acting as templates for areverse transcription reaction in the presence of compatible primers.Primers may be appended according to techniques well known to thoseskilled in the art by means of an enzyme, such as for example RNA ligasederived from phage T4 and which catalyzes intermolecular phosphodiesterbond formation between a 5′ phosphate group of a donor molecule and a 3′hydroxyl group of an acceptor molecule. Such an RNA ligase iscommercially available (for example Life Technologies—GIBCO BRL catalognumber 18003). The cDNAs thus obtained may then be amplified byconventional techniques (PCR for example) using the appropriate primers,as illustrated in FIG. 3. This technique is especially adapted tocloning short RNA molecules (less than 1000 bases).

Another approach for cloning and/or identifying specific RNA regionsinvolves for example a reverse transcription reaction, performed uponthe digests of an enzyme acting specifically on double stranded RNA,such as RNase H, using random primers, which will randomly initiatetranscription along RNAs. cDNAs thus obtained are then amplifiedaccording to conventional molecular biology techniques, for example byPCR using primers formed by appending oligonucleotides to cDNA ends bymeans of T4 phage DNA ligase (commercially available; for example fromLife Technologies—GIBCO BRL catalog number 18003). This second techniqueis illustrated in FIG. 4 and in the examples. This technique isespecially adapted to long RNAs, and provides a sufficient part of thesequence data to subsequently reconstruct the entire initial sequence.

A further approach for cloning and/or identifying specific RNA regionsis equally based on a reverse transcription reaction using randomprimers (FIG. 4). However, according to this variant, the primers usedare at least in part semi-random primers, i.e. oligonucleotidescomprising:

-   -   a random (degenerated) region,    -   a minimal priming region having a defined degree of constraint,        and    -   a stabilizing region.

Preferably, these are oligonucleotides comprising, in the 5′→3′direction:

-   -   a stabilizing region comprising 8 to 24 defined nucleotides,        preferably 10 to 18 nucleotides. This stabilizing region may        itself correspond to the sequence of an oligonucleotide used to        reamplify fragments derived from initial amplifications        performed by means of the semi-random primers of the invention.        In addition, the stabilizing region may comprise the sequence of        one or more sites, preferably non-palindromic, corresponding to        restriction enzymes. This makes it possible for example to        simplify the cloning of the fragments thus amplified. A        particular example of a stabilizing region is given by the        sequence GAG AA CGT TAT (residues 1 to 12 of SEQ ID NO:1);    -   a random region having 3 to 8 nucleotides, more particularly 5        to 7 nucleotides, and    -   a minimal priming region defined such that the oligonucleotide        hybridizes on average at least about every 60 base pairs,        preferably about every 250 base pairs. More preferentially. the        priming region comprises 2 to 4 defined nucleotides, preferably        3 or 4, such as for example AGGX, where X is one of the four        bases A, C, G or T. The presence of such a priming region gives        the oligonucleotide the capacity to hybridize on average about        every 256 base pairs.

In an especially preferential manner, the oligonucleotides have theformula:

GAGAAGCGTTATNNNNNNNAGGX (SEQ ID NO: 1) where the fixed bases are orderedso as to minimize background due to self-pairing in PCR experiments,where N indicates that the four bases may be present in a random fashionat the indicated position, and where X is one of the four bases A, C, Gor T. Such oligonucleotides equally constitute an object of the presentinvention.

In this respect, so as to increase the priming events on the RNAs to becloned, reactions may be carried out in parallel with oligonucleotidessuch as:GAGAAGCGTTATNNNNNNNAGGT (oligonucleotides A)  (SEQ ID NO: 1; X=T)GAGAAGCGTTATNNNNNNNAGGA (oligonucleotides B)  (SEQ ID NO: 1; X=A)GAGAAGCGTTATNNNNNNNAGGC (oligonucleotides C)  (SEQ ID NO: 1; X=C),GAGAAGCGTTATNNNNNNNAGGG (oligonucleotides D)  (SEQ ID NO: 1; X=G)each oligonucleotide population (A, B, C, D) being able to be used aloneor in combination with another.

After the reverse transcription reaction, the cDNAs are amplified by PCRusing oligonucleotides A or B or C or D.

As indicated hereinabove, depending on the complexity and thespecificity of the desired oligonucleotide population, the number ofdegenerated positions may range from 3 to 8, preferably from 5 to 7.Below 3 hybridizations are limited and above 8 the oligonucleotidepopulation is too complex to ensure good amplification of specificbands.

Furthermore, the length of the fixed 3′ end (constrained priming region)of these oligonucleotides may also be modified: while the primersdescribed above, with 4 fixed bases, allow amplification of 256 basepair fragments on average, primers with 3 fixed bases allowamplification of shorter fragments (64 base pairs on average). In afirst preferred embodiment of the invention, one uses oligonucleouidesin which the priming region comprises 4 fixed bases. In anotherpreferred embodiment of the invention, one uses oligonucleotides havinga priming region of 3 fixed bases. In fact, as exons have an averagesize of 137 bases, they are advantageously amplified with sucholigonucleotides. In this respect, refer also to oligonucleotides withsequence SEQ ID NO: 2, 3 and 4, for example.

Finally, in general, the identification and/or cloning step of RNA isbased on different methods of PCR and cloning, so as to generate as muchinformation as possible.

Identification and/or cloning starting with heterotriplexes.

In the case of heterotriplex structures (another variant of the method),the qualitatively different regions (insertions, deletions, differentialsplicing) appear essentially in the form of double stranded DNA regions,as shown in FIG. 5. Such regions may thus be identified and cloned bytreating them in the presence of appropriate enzymes such as an enzymecapable of digesting RNA, and next by an enzyme capable of digestingsingle stranded DNA. The nucleic acids are thus directly obtained in theform of double stranded DNA and can be cloned into any suitable vector,such as the vector pMos-Blue (Amersham, RPN 5110), for example. Thismethodology should be distinguished from previously described approachesusing RNAs or oligonucleotides of predetermined sequences, modified soas to have nuclease activity (Landgraf et al., (1994), Biochemistry, 33:10607-10615).

Identification and/or cloning starting with DNA/DNA homoduplexes (FIG.6).

The fragments isolated on the basis of their atypical structures arethen ligated, at each of their ends, to adaptors, or linkers, havingcleaved restriction sites at one of their ends. This step may be carriedout according to the techniques known to those skilled in the art, forexample by ligation with phage T4 DNA ligase. The restriction sites thusintroduced are chosen to be compatible with the sites of the cDNAfragments. The linkers introduced are double stranded cDNA sequences, ofknown sequence, making it possible to generate the primers for enzymaticamplifications (PCR). Since the next step consists in amplifying the twostrands which each bear the qualitative differences to be identified, itis necessary to use linkers with phosphorylated 5′ ends. Thus after heatdenaturation of double stranded cDNA appended with linkers, each ofthese cDNA ends is covalently linked to a specific priming sequence.Following PCR by means of appropriate specific primers, two categoriesof double stranded cDNA are obtained: fragments which contain sequencesspecific of qualitative differences which distinguish the twopathophysiological conditions, and fragments which comprise the negativefingerprint of these splicing events. Cloning these fragments generatesan alternative splicing library in which, for each splicing event,positive and negative fingerprints are present. This library thereforegives access not only to alternative exons and introns but also to thespecific junctions formed by excision of these spliced sequences. In asame library, this differential genetic information may be derived fromtwo pathophysiological conditions indiscriminately. Furthermore, so asto check the differential nature of the identified splicing events andso as to determine the condition in which they are specificallyelicited, the clones in the library may be hybridized with probesderived from each of the total mRNA populations.

The cDNA fragments derived from the qualitative differences soidentified have two principal uses:

-   -   cloning into suitable vectors so as to construct libraries        representative of the qualitative differences occurring between        the two pathophysiological conditions under study,    -   use as probes to screen a DNA library allowing identification of        differential splicing events.

The vectors used in the invention can be in particular plasmids,cosmids, phages, YAC, HAC, etc. These nucleic acids may thus be storedas such, or introduced into microorganisms compatible with the cloningvector being used, for replication and/or stored in the form ofcultures.

The time interval required for carrying out the methods herein describedfor each sample is generally less than two months, in particular lessthan 6 weeks. Furthermore, these different methods may be automated sothat the total length of time is reduced and treatment of a large numberof samples is simplified.

In this regard, another object of the invention concerns nucleic acidsthat have been identified and/or cloned by the methods of the invention.As already noted, these nucleic acids may be RNAs or cDNAs. Moregenerally, the invention concerns a nucleic acid composition,essentially comprising nucleic acids corresponding to alternativesplicings which are distinctive of two physiological conditions. Moreparticularly, these nucleic acids correspond to alternative splicingsidentified in a biological test sample and not present in the samebiological sample under a reference condition. The invention is equallyconcerned with the use of the nucleic acids thus cloned as therapeuticor diagnostic products, or as screening tools to identify activemolecules, as set forth hereinafter.

The different methods disclosed hereinabove thus all lead to the cloningof cDNA sequences representative of differentially spliced geneticinformation between two pathophysiological conditions. The whole set ofclones derived from one of these methods makes it thus possible toconstruct a library representative of qualitative differences occurringbetween two conditions of interest.

Generation of Qualitative Libraries

In this respect, the invention is further directed to a method forpreparing nucleic acid libraries representative of a given physiologicalstate of a biological sample. This method advantageously comprisescloning nucleic acids representative of qualitative markers of geneticexpression (for example alternative splicings) of said physiologicalstate but not present in a reference state, to generate librariesspecific to qualitative differences occurring between the two statesbeing investigated.

These libraries are constituted by cDNA inserted in plasmid or phagevectors. Such libraries can be deposited on nitrocellulose filters orany other support known to those skilled in the art, such as chips orbiochips.

One of the features as well as one of the original characteristics ofqualitative differential screening is that this technique leads not toone but advantageously to two differential libraries which represent thewhole set of qualitative differences occurring between two givenconditions: a library pair (see FIG. 1D).

Thus, the invention preferentially concerns any nucleic acid compositionor library that can be obtained by hybridizing RNAs derived from a firstbiological sample with cDNAs derived from a second biological sample.More preferentially, the libraries or compositions of the inventioncomprise nucleic acids representative of qualitative differences inexpression between two biological samples, and are generated by a methodcomprising (i) at least one hybridization step between RNAs derived froma first biological sample and cDNAs derived from a second biologicalsample, (ii) selecting those nucleic acids representative of qualitativedifferences in expression and, optionally, (iii) cloning said nucleicacids.

Furthermore, once such libraries are constructed, it is possible toproceed with a step of clone selection in order to improve thespecificity of the resulting libraries. Indeed, it may be that certainmismatches observed are not due solely to qualitative differences (eg.,to differential alternative splicings) but might result from reversetranscription defects for example. Although such events are notgenerally significant, it is preferable to prevent them or reduce theirincidence prior to nucleic acid cloning. To accomplish this, the libraryclones may be hybridized with the cDNA populations occurring in bothphysiological conditions being investigated (cf. step © hereinabove).The clones which hybridize in a non-differential manner with bothpopulations would be considered as nonspecific and optionally discardedor treated as second priority (in fact, the appearance of a new isoformin the test sample does not always indicate that the initial isoformpresent in the reference sample has disappeared from this test sample).Clones hybridizing with only one of either populations or hybridizingpreferentially with one of the populations are considered specific andcould be selected in priority to constitute enriched or refinedlibraries.

A refining step may be equally performed by hybridizing and checking theidentify of clones by means of probes derived from a statisticallyrelevant number of pathological samples.

The present application is therefore equally directed to any nucleicacid library comprising nucleic acids specific to alternative splicingstypical of a physiological condition. These libraries advantageouslycomprise cDNAs, generally double stranded, corresponding to RNA regionsspecific of alternative splicing. Such libraries may be comprised ofnucleic acids, generally incorporated within a cloning vector, or ofcell cultures containing said nucleic acids.

The choice of initial RNAs partly determines the characteristics of theresulting libraries:

-   -   the RNAs of both conditions A and B are mRNAs or total mature        RNAs isolated according to techniques known to those skilled in        the art. The libraries are thus so-called restricted qualitative        differential screening libraries, since they are restricted to        qualitative differences that characterize the mature RNAs of        both pathophysiological conditions.    -   the RNAs of one of either conditions are mRNAs or mature total        RNAs whereas the RNAs of the other condition are premessenger        RNAs, not processed by splicing, isolated according to        techniques known to those skilled in the art, from cell nuclei.        In this situation the resulting libraries are so-called complex        differential screening libraries, as being not restricted to        differences between mature RNAs but rather comprising the whole        set of spliced transcripts in a given condition which are absent        from the other, including all introns.    -   finally, the RNAs could arise from a single pathophysiological        condition and in this case the differential screening involves        mature RNAs and premessenger RNAs of the same sample. In such a        case, the resulting libraries are autologous qualitative        differential screening libraries. The usefulness of such        libraries lies in that they include exclusively the whole range        of introns transcribed in a given condition. Whether they        hybridize with a probe derived from mature RNAs of a distinct        condition allows one to quickly ascertain if the condition under        study is characterized by persisting introns while providing for        their easy identification.

Generally speaking, the libraries are generated by spreading, on a solidmedium (notably on agar medium), of a cell culture transformed by thecloned nucleic acids. Transformation is done by any technique known tothose skilled in the art (transfection, calcum phosphate precipitation,electroporation, infection with bacteriophage, etc.). The cell cultureis generally a bacterial culture, such as for example E. coli. It mayalso be a eukaryotic cell culture, notably lower eukaroytic cells(yeasts for example). This spreading step can be performed in sterileconditions on a dish or any other suitable support. Additionally, thespread cultures on agar medium can be stored in a frozen state forexample (in glyerol or any other suitable agent). Naturally, theselibraries can be used to produce “duplicates”, i.e. copies madeaccording to common techniques more fully described hereinafter.Furthermore, such libraries are generally used to prepare an amplifiedlibrary, i.e. a library comprising each clone in an amplified state. Anamplified library is prepared as follows: starting from a spreadculture, all cellular clones are recovered and packaged for storage inthe frozen state or in a cold place, using any compatible medium. Thisamplified library is advantageously prepared from E. coli bacterialcultures, and is stored at 4° C., in sterile conditions. This amplifiedlibrary allows preparation and unlimited replication of any subsequentlyprepared library containing such clones, on different supports, for avariety of applications. Such a library further allows the isolation andcharacterization of any clone of interest. Each clone composing thelibraries of the invention is indeed a characteristic element of aphysiological condition, and constitutes therefore a particularlyinteresting target for various studies such as the search for markers,antibody production, diagnostics, gene transfer therapy, etc. Thesedifferent applications are discussed in more detail below. The libraryis generally prepared as described above by spreading the cultures in anagar medium, on a suitable support (petri dish for example). Theadvantage of using an agar medium is that each colony can be separatedand distinctly recognized. Starting from this culture, identicalduplicates may be prepared in substantial amounts simply byreplica-plating on any suitable support according to techniques known inthe art. Thus, the duplicate may be obtained by means of filters,membranes (nylon, nitrocellulose, etc.) on which cell adhesion ispossible. Filters may then be stored as such, at 4° C. for example, in adried state, in any packing medium that does not after nucleic acids.Filters may equally be treated in such a manner as to discard cells,proteins, etc., and to retain only such components as nucleic acids.These treatment procedures may notably comprise the use of proteases,detergents, etc. Treated filters may be equally stored in any device orunder any condition acceptable for nucleic acids.

The nucleic acid libraries can be equally directly prepared from nucleicacids, by transfer onto biochips or any other suitable device.

The invention is equally directed to any library comprisingoligonucleotides specific of alternative splicing events thatdistinguish two physiological conditions. These are advantageouslysingle stranded oligonucleotides comprising from 5 to 100-mers,preferably less than 50-mers, for example in the range of 25-mers.

These oligonucleotides are specific of alternative splicingsrepresentative of a given condition or type of physiological condition.Thus, such oligonucleotides may for example be oligonucleotidesrepresentative of alternative splicing events characteristic ofapoptotic states. Indeed, it has been reported in the literature thatcertain alternative splicing events are observed in apoptoticconditions. This holds especially true for splicing within Bclx, Bax,Fas or Grb2 genes for example. By referring to published data orsequences available in the literature and/or in databases, it ispossible to generate oligonucleotides specific to spliced or unsplicedforms. These oligonucleotides may for example be generated according tothe following strategy:

(a) identifying a protein or a splicing event characteristic of anapoptotic condition and the sequence of the spliced domain. Thisidentification procedure can be based upon published data or acompilation of available sequences in databases;

(b) synthesizing artificially one or more oligonucleotides correspondingto one or more regions of this domain, which therefore allow theidentification of the unspliced form in the RNAs of a test samplethrough hybridization;

(c) synthesizing artificially one or more oligonucleotides correspondingto the junction region between two domains separated by the spliceddomain. These oligonucleotides therefore allow the identification of thespliced form in the RNAs of a test sample through hybridization;

(d) repeating steps (a) to (c) listed above with other proteins orsplicing events characteristic of apoptotic conditions;

(e) transferring upon a first suitable support one or a plurality ofoligonucdeotides specific to apoptotic forms of messengers identifiedhereinabove and, upon another suitable support, one or a plurality ofoligonucleotides specific to non-apoptotic forms.

The two supports thus obtained may be used to assess the physiologicalstate of cells or test samples, and particularly their apoptotic state,through hybridization of a nucleic acid preparation derived from suchcells or samples.

Other similar libraries can be generated using oligonucleotides specificto different pathophysiological states (neurodegeneration, toxicity,proliferation, etc.), thus broadening the range of applications.

Alternative intron or exon libraries can also be in the form ofcomputerized data base systems compiled by systematically analyzingdatabases in which information about genomes of individual organisms,tissues or cell cultures is recorded. In such a case, the data obtainedby elaboration of such virtual databases may be used to generateoligonucleotide primers that will serve in testing twopathophysiological conditions in parallel.

The computerized databases may further be used to derive versatilenucleotide probes, representative of a given class of proteins, orspecific of a particular sequence. These probes can then be deposited onthe clone libraries derived from different alternative intron and exoncloning techniques in order to appreciate the complexity of thesemolecular libraries and rapidly determine whether a given class ofprotein or a given defined sequence is differentially spliced whencomparing two distinct pathophysiological states.

A further nucleic acid composition or library according to the inventionis an antisense library, generated from the sequences identifiedaccording to the methods of the invention (DATAS). To generate this typeof library, such sequences are cloned so as to be expressed as RNAfragments corresponding to an antisense orientation relative to themessenger RNAs used for DATAS. This results in a so-called antisenselibrary. This approach preferentially makes use of the cloning variantwhich allows orientation of the cloned fragments. The usefulness of suchan antisense library is that it allows transfection of cell lines andmonitoring of all phenotypic alterations whether morphological orenzymatic, or revealed by the use of reporter genes or genes that conferresistance to a selective agent. Analysis of phenotypic variationssubsequent to the introduction of an antisense expression vector isgenerally done after selection of so-called stable clones, i.e. allowingcoordinated replication of the expression vector and the host genome.This coordination is enabled through the integration of the expressionvector into the cellular genome or, when the expression vector isepisomal, through selective pressure. Such selective pressure is appliedby treating the transfected cell culture with a toxic agent that canonly be detoxified when the product of a gene carried by the expressionvector is expressed within the cell. This results in synchronizationbetween host and transgene replication. One advantageously uses episomalvectors derived from the Epstein-Barr virus which allow expression of 50to. 100 vector copies within a given cell (Deiss et al., (1996), EMBOJ., 15: 3861-3870; Kissil et al., (1995), J. Biol. Chem, 270:27932-27936).

The advantage of these antisense libraries related to the DATASsequences they contain is that they not only allow identification of thegene the expression of which is inhibited to produce the selectedphenotype, but also identification of which splicing isoform of thisgene was affected. When the antisense fragment targets a given exon, itmay be deduced therefrom that the protein domain and thus the functioninvolving this domain counteracts the observed phenotype. In thisrespect coupling of DATAS with an antisense approach represents ashortcut towards functional genomics.

DNA Chips

The invention is further directed to any support material (membrane,filter, biochip, chip, etc.) comprising a nucleic acid composition orlibrary as defined hereinabove. This may more particularly be a celllibrary or a nucleic acid library. The invention also concerns any kitor support material comprising several libraries according to theinvention. In particular, it may be advantageous to use in parallel alibrary representative of the qualitative features of a testphysiological condition with respect to a reference physiologicalcondition and, as control, a library representative of the features of areference physiological condition in relation to the test physiologicalcondition (a “library pair”). An advantageous kit according to theinvention thus comprises two differential qualitative librariesbelonging to two physiological conditions (a “library pair”). Accordingto one particular embodiment, the kits pursuant to the inventioncomprise several library pairs as defined hereinabove, corresponding todistinct physiological states or to different biological samples forexample. The kits may comprise for example these different library pairsarranged serially on a common support.

Generation of Probes

Another use of the cDNA compositions according to the invention,representative of qualitative differences occurring between twopathophysiological states, consists in deriving probes thereof. Suchprobes may in fact be used to screen differential splicing eventsbetween two pathophysiological conditions.

These probes (see FIG. 1D) may be prepared by labeling nucleic acidlibraries or populations according to conventional techniques known inthe art. Thus, the labeling may be carried out by enzymatic,radioactive, fluorescent, immunological means, etc. The labeling ispreferably radioactive or fluorescent. This type of labeling may beaccomplished for example by introducing into the nucleic acid population(either after synthesis or during synthesis) labeled nucleotides,enabling their visualization by conventional methods.

One application is therefore to screen a conventional genomic library.Such a library may comprise, depending on whether the vector is derivedfrom a phage or a cosmid, DNA fragments of 10 kb to 40 kb. The number ofclones hybridizing with the probes generated by DATAS and representativeof differential splicing events occurring between two conditions thusapproximately reflects the number of genes affected by alternativesplicings, according to whether they are expressed in one or the othercondition being investigated.

Preferably, the probes of the invention are used to screen a genomic DNAlibrary (generally of human origin) adapted to identifying splicingevents. Such a genomic library is preferably composed of DNA fragmentsof restricted size (generally cloned into vectors), so as to yieldstatistically only a single differentially spliceable element, i.e. asingle exon or a single exon. The genomic DNA library is thereforeprepared by digesting genomic DNA with an enzyme having a recognitionsite restricted by 4 bases, thus providing the possibility of obtainingby controlled digestion DNA fragments with an average size of 1 kb. Suchfragments require the generation of 10⁷ clones to constitute a DNAlibrary representative of a higher eukaryotic genome. Such a library isequally an object of the present application. This library is thenhybridized with the probes derived from qualitative differentialscreening. In fact, for each experiment being investigated and whichcompares two pathophysiological conditions A and B, two probes (probepair) are obtained. One probe is enriched in splicing eventscharacteristic of condition A and one probe is enriched in splicingmarkers characteristic of B. Clones in the genomic library whichhybridize preferentially with one of either probe harbor sequences thatare preferentially spliced in the corresponding pathophysiologicalconditions.

The methods of the invention thus provide for the systematicidentification of qualitative differences in gene expression. Thesemethods have many applications, related to the identification and/orcloning of molecules of interest, in the fields of toxicology,pharmacology or still, in pharmacogenomics for example.

Applications

The invention is therefore additionally concerned with the use of themethods, nucleic acids or libraries previously described for identifyingmolecules of therapeutic or diagnostic value. The invention is morespecifically concerned with the use of the methods, nucleic acids orlibraries described hereinabove for identifying proteins or proteindomains that are altered in a pathology.

One of the major strengths of these techniques is, indeed, theidentification, within a messenger, and consequently within thecorresponding protein, of the functional domains which are affected in agiven disorder. This makes it possible to assess the importance of agiven domain in the development and persistence of a pathological state.The direct advantage of restricting to a given protein domain the impactof a pathological disorder resides in that the latter can be viewed as arelevant target for screening small molecules for therapeutic purposes.This information further constitutes a key for designing therapeuticallyactive polypeptides that may be delivered by gene therapy; suchpolypeptides can notably be single chain antibodies derived fromneutralizing antibodies directed against domains identified by thetechniques herein described.

More specifically, the methods according to the invention providemolecules which:

-   -   may be coding sequences derived from alternative exons.    -   may correspond to noncoding sequences borne by introns        differentially spliced between two pathophysiological states.

From these two points, different information can be obtained.

Alternative splicings of exons which discriminate between twopathophysiological states reflect a regulatory mechanism of geneexpression capable of modulating (in more precise terms suppressing orrestoring) one or a number of functions of a particular protein.Therefore, as the majority of structural and functional domains (SH2,SH3, PTB, PDZ, and catalytic domains of various enzymes) are encoded byseveral contiguous exons, two configurations might be considered:

i) the domains are truncated in the pathological condition (Zhu, Q. etal., (1994), J. Exp. Med., 180 (2): 461-470); this indicates that thesignaling pathways involving such domains must be restored fortherapeutical purposes.

ii) the domains are retained in the course of a pathological disorderwhereas they are absent in the healthy state; these domains can beconsidered as screening targets for low molecular weight compoundsintended to antagonize signal transduction mediated by such domains.

The differentially spliced sequences may correspond to noncoding regionslocated 5′ or 3′ of the coding sequence or to introns occurring betweentwo coding exons. In the noncoding regions, these differential splicingscould reflect a modification of messenger stability or translatability(Bloom, T. J. and Beavo, J. A., (1995), Proc. Natl. Acad. Sci. USA, 93(24): 14188-14192; Ambartsumian, N. et al., (1995), Gene, 159 (1):125-130). A search for these phenomena should be conducted based on suchinformation and might qualify the corresponding protein as a candidatetarget in view of its accumulation or disappearance. Retention of anintron in a coding sequence often results in the truncation of thenative protein by introducing a stop codon within the reading frame(Varesco, L., et al., (1994). Hum. Genet., 93 (3): 281-286; Canton, H.,et al., (1996), Mol. Pharmacol., 50 (4): 799-807; Ion, A., et al.,(1996), Am. J. Hum. Genet., 58 (6): 1185-1191). Before such a stop codonis read, there generally occurs translation of a number of additionalcodons whereby a specific sequence is appended to the translatedportion, which behaves as a protein marker of alterative splicing. Theseadditional amino acids can be used to produce antibodies specific to thealternative form inherent to the pathological condition. Theseantibodies may subsequently be used as diagnostic tools. The truncatedprotein undergoes a change or even an alteration in properties. Thusenzymes may loose their catalytic or regulatory domain. becominginactive or constitutively activated. Adaptors may lose their capacityto link different partners of a signal transduction cascade (Watanabe,K. et al., (1995), J. Biol. Chem., 270 (23): 13733-13739). Splicingproducts of receptors may lead to the formation of receptors having losttheir ability to bind corresponding ligands (Nakajima, T. et al.,(1996), Life Sci., 58 (9): 761-768) and may also generate soluble formsof receptor by release of their extracellular domain (Cheng J., (1994),Science, 263 (5154): 1759-1762). In this case, diagnostic tests can bedesigned, based on the presence of circulating soluble forms of receptorwhich bind a given ligand in different physiological fluids.

The invention is more specifically concerned with the use of themethods, nucleic acids or libraries described hereinabove foridentifying antigenic domains that are specific for proteins involved ina pathology. The invention is equally directed to the use of the nucleicacids, proteins or peptides as described above for diagnosingpathological conditions.

The invention is equally directed to a method for identifying and/orproducing proteins or protein domains involved in a pathologycomprising:

(a) hybridizing messenger RNAs of a pathological sample with cDNAs of ahealthy sample, or vice versa, or both in parallel,

(b) identifying, within the hybrids formed, regions corresponding toqualitative differences (unpaired (RNA) or paired (double stranded DNA))which are specific to the pathological state in relation to the healthystate,

(c) identifying and/or producing the protein or protein domaincorresponding to one or several regions identified in step (b).

The regions so identified generally correspond to differentialsplicings, but they may also correspond to other genetic alterationssuch as insertion(s) or deletion(s), for example.

The protein(s) or protein domains may be isolated, sequenced, and usedin therapeutic or diagnostic applications, notably for antibodyproduction.

To better illustrate this point, the qualitative differential screeningof the invention allows one to advantageously identify tumor suppressorgenes. Indeed, may examples indicate that one way suppressor genes areinactivated in the course of tumor progression is inactivation bymodulation of alternative forms of splicing.

Hence, in small cell lung carcinoma, the gene of protein p130 belongingto the RB family (retinoblastoma protein) is mutated at a consensussplicing site. This mutation results in the removal of exon 2 and in theabsence of synthesis of the protein due to the presence of a prematurestop codon. This observation was the first of its kind to underscore theimportance of RB family members in tumorigenesis. Likewise, in certainnon small cell lung cancers, the gene of protein p161NK4A, a proteinwhich is an inhibitor of cyclin-dependent kinases cdk4 and cdk6, ismutated at a donor splicing site. This mutation results in theproduction of a truncated protein with a short half-life, leading to theaccumulation of the inactive phosphorylated forms of RB. Furthermore,WT1, the Wilm's tumor suppressor gene, is transcribed into severalmessenger RNAs generated by alternative splicings. In breast cancers,the relative proportions of different variants are modified incomparison to healthy tissue, thereby yielding diagnostic tools or cluesto understanding the importance of the various functional domains of WT1in tumor progression. The same alteration process affecting ratiosbetween different messenger RNA forms and protein isoforms duringcellular transformation is again found in the case of neurofibrin NF1.In addition, the concept that modulation of splicing phenomena behavesas a marker of tumor progression is further supported by the example ofHDM2 where five alternative splicing events are detected in ovarian andpancreatic carcinoma, the expression of which increases depending on thestage of tumor development. Furthermore, in head and neck cancers, oneof the mechanisms by which p53 is inactivated involves a mutation at aconsensus splicing site.

These few examples clearly illustrate the interest of the methods of theinvention based on systematic screening for alternative splicingpatterns which discriminate between a given tumor and an adjacenthealthy tissue. Results thus obtained allow not only thecharacterization of known tumor suppressor genes but also, in view ofthe original and systematic aspect of qualitative differential screeningmethods, the identification of novel alternative splicings specific totumors that are likely to affect new tumor suppressor genes.

The invention is therefore further directed to identifying and/orcloning tumor suppressor genes or genetic alterations (eg., splicingevents) within those tumor suppressor genes, as previously defined. Thismethod may advantageously comprise the following steps:

(a) hybridizing messenger RNAs of a tumor sample with cDNAs of a healthysample, or vice versa, or both in parallel,

(b) identifying, within the hybrids formed, regions specific to thetumor sample in relation to the healthy sample,

(c) identifying and/or cloning the protein or protein domaincorresponding to one or more regions identified in step (b).

The tumor suppressor properties of the proteins or protein domainsidentified may then be tested in different known models. These proteins,or their native forms (displaying the splicing pattern observed inhealthy tissue) may then be use for various therapeutic or diagnosticapplications, notably for antitumoral gene therapy.

The present application therefore relates not only to different aspectsof embodying the present technology but also to the exploitation of theresulting information in research, development of screening assays forchemical compounds of low molecular weight, and development of genetherapy or diagnostic tools.

In this connection, the invention further concerns the use of themethods, nucleic acids or libraries described above in genotoxicology,i.e. to predict the toxicity of test compounds.

The genetic programs initiated during treatment of cells or tissues bytoxic agents are predominantly correlated with apoptotic processes, orprogrammed cell death. The importance of alternative splicing processesin regulating such apoptotic mechanisms is well described in theliterature. However, no single gene engineering technique described todate allows exhaustive screening and isolation of sequence variationsdue to alternative splicings distinctive of two given pathophysiologicalconditions. The qualitative differential splicing screening methodsdeveloped by the present invention make it possible to gather allsplicing differences occurring between two conditions within cDNAlibraries. Comparing RNA sequences (for example messenger RNAs) of atissue (or of a cell culture) either treated or not with a standardtoxic compound allows the generation of cDNA libraries which comprisegene expression qualitative differences characterizing the toxic effectbeing investigated. These cDNA libraries may then be hybridized withprobes derived from RNA arising from the same tissues or cells treatedwith the chemical being assessed for toxicity. The relative capacity ofthese probes to hybridize with the genetic sequences specific to a givenstandard toxic condition allows toxicity of the compound to bedetermined. Furthermore, in addition to the use of DATAS for thegeneration and utilization of qualitative differential libraries inducedby toxic agents, a part of the invention consists equally indemonstrating that regulation defects in the splicing of certainmessenger RNAs may be induced by certain toxic agents, at doses lowerthan the IC50 determined in the cytotoxicity and apoptosis tests knownto those skilled in the art. Such regulation defects (or deregulations)may be used as markers to assess the toxicity and/or potency ofmolecules (chemical or genetic).

The invention therefore equally concerns any method for detecting ormonitoring the toxicity and/or therapeutic potential of a compound basedon the detection of splicing forms and/or patterns induced by thiscompound on a biological sample. It further concerns the use of anymodification of splicing forms and/or patterns as a marker to assess thetoxicity and/or potency of molecules.

Toxicity assessment or monitoring may be performed more specificallyfollowing two approaches:

According to a first approach, the qualitative differential screeningmay be accomplished between a reference tissue or cell culture notsubjected to treatment on the one hand, and treated by the product whosetoxicity is to be assessed on the other hand. The analysis of clonesrepresentative of qualitative differences specifically induced by thisproduct subsequently provides for the eventual detection within theseclones of events closely related to cDNA involved in toxic reactionssuch as apoptosis.

Such markers are monitored as they arise as a function of the dose andduration of treatment by the product in question so that thetoxicological profile thereof may be established.

The present application is therefore equally directed to a method foridentifying, by means of qualitative differential screening according tothe methods set forth above, toxicity markers induced in a modelbiological system by a chemical compound whose toxicity is to bemeasured. In this respect, the invention relates in particular to amethod for identifying and/or cloning nucleic acids specific of a toxicstate of a given biological sample comprising preparing qualitativedifferential libraries between the cDNAs and the RNAs of the sampleeither subjected or not to treatment by the test toxic compound, andsearching for toxicity markers specific to the properties of the samplepost-treatment.

According to the second approach, abacus are prepared for differentclasses of toxic products, that are fully representative of the toxicityprofiles as a function of dosage and treatment duration for a givenreference tissue or cell model. For each abacus dot, cDNA librariesrepresentative of qualitative genetic differences can be generated. Thelatter represent qualitative differential libraries, i.e. they areobtained by extracting genetic information from the dot selected in theabacus diagram and from the corresponding dot in the control tissue orcell model. As set forth in the examples, the qualitative differentialscreening is based on hybridizing mRNA derived from one condition withcDNAs derived from another condition. As noted above, the qualitativedifferential screening may also be conducted using total RNAs or nuclearRNAs containing premessenger species.

In this respect, the invention concerns a method for determining orassessing the toxicity of a test compound to a given biological samplecomprising hybridizing:

-   -   differential libraries between cDNAs and RNAs of said biological        sample from a healthy state and at various stages of toxicity        resulting from treatment of said sample with a reference toxic        compound, with,    -   a nucleic acid preparation of the biological sample treated by        said test compound, and    -   assessing the toxicity of the test compound by determining the        extent of hybridization with the different libraries.

According to this method, it is advantageous to proceed with two crosshybridizations for each condition (compound dosage and/or incubationtime), between:

-   -   RNAs from condition A (test) and cDNAs from condition B        (reference) (rA/cB)    -   RNAs from condition B (reference) and cDNAs from condition A        (test) (rB/cA).

Each reference toxic condition, at each abacus dot, thus corresponds totwo qualitative differential screening libraries. One or such librariesis a full collection of qualitative differences, i.e. notably thealternative splicing events, specific to the normal reference conditionwhereas the other library is a full collection of,splicing eventsspecific to the toxic situations. These libraries are replica-plated onsolid support materials such as nylon or nitrocellulose filters oradvantageously on chips. These libraries initially formed of cDNAfragments of variable length (according to the splicing events beingconsidered) may be optimized by using oligonucleotides derived frompreviously isolated sequences.

Where a chemical compound is a candidate for pharmaceutical development,this may be tested with the same tissue or cell models as those recordedin the toxicity abacus diagram. Molecular probes may then be synthesizedfrom mRNAs extracted from the biological samples treated with thechemical compound of interest. These probes are then hybridized onfilters bearing cDNA of rA/cB and rB/cA libraries. For instance, therA/cB library may contain sequences specific to the normal condition andthe rB/cA library may contain alternative spliced species specific tothe toxic condition. Innocuity or toxicity of the chemical compound isthen readily assessed by examining the hybridization profile of anmRNA-derived probe belonging to the reference tissue or cell model thathas been treated by the test compound:

-   -   efficient hybridization with the rA/cB library and no signal in        the rB/cA library demonstrates that the compound has no toxicity        in the model under study    -   positive hybridization between the probe and the rB/cA library        dones is evidence of test compound-induced toxicity.

Practical applications related to such libraries may be provided byhepatocyte culture models, such as the HepG2 line, renal epithelialcells, such as the HK-2 line, or endothelial cells, such as the ECV304line, following treatment by toxic agents such as ethanol, camptothecinor PMA.

A preferred example may be provided by use in cosmetic testing of skinculture models subjected or not to treatment by toxic agents orirritants.

A further object of the present application is therefore differentialscreening libraries (between cDNAs and RNAS) made from reference organs,tissues or cell cultures treated by chemical compounds representative ofbroad classes of toxic agents according to abacus charts disclosed inthe literature. The invention further encompasses the spreading of theselibraries on filters or support materials known to those skilled in theart (nitrocellulose, nylon . . . ). Advantageously, these supportmaterials may be chips which hence define genotoxicity chips. Theinvention is further concerned with the potential exploitation of thesequencing data about different clones making up these libraries inorder to understand the mechanisms underlying the action of varioustoxic agents, as well as with the use of such libraries in hybridizationwith probes derived from cells or tissues treated by a chemical compoundor a pharmaceutical product whose toxicity is to be determined.Advantageously, the invention relates to nucleic acid libraries such asof the type defined above, prepared from skin cells treated underdifferent toxic conditions. The invention is further concerned with akit comprising these individual skin differential libraries.

The invention is further directed to the use of the methods, nucleicacids or libraries previously described to assess (predict) or enhancethe therapeutic effectiveness of test compounds (genopharmacology).

In this particular use, the underlying principle is very similar to thatpreviously described. Reference differential libraries are establishedbetween cDNAs and RNA from a control cell culture of organ andcounterparts thereof simulating a pathological model. The therapeuticefficacy of a product may then be evaluated by monitoring its potentialto antagonize qualitative variations of gene expression which arespecific of the pathological model. This is demonstrated by a change inthe hybridization profile of a probe derived from the pathological modelwith the reference libraries: in the absence of treatment, the probeonly hybridizes with the library containing the specific markers of thedisease. Following treatment with an effective product, the probe,though it is derived from the pathological model, hybridizespreferentially with the other library, which bears the markers of thehealthy model equivalent.

In this respect, the model is further directed to a method fordetermining or assessing the therapeutic efficacy of a test compound ona given biological sample comprising hybridizing:

-   -   differential libraries between cDNAs and RNAs from said        biological sample in a healthy state and in a pathological state        (at different development stages), with,    -   a preparation of nucleic acids derived from the biological        sample treated by said test compound, and    -   assessing the therapeutic potential of the test compound by        determining the extent of hybridization with the different        libraries.

Such an application is exemplified by an apoptosis model simulatingcertain aspects of neurodegeneration which are antagonized by standardtrophic factors. Thus, cells derived from the PC12 pheochromocytoma linewhich differentiate into neurites in the presence of NGF enter intoapoptosis upon removal of this growth factor. This apoptotic process isaccompanied by expression of many programmed cell death markers. severalof which are regulated by alternative splicing and downregulated byIGF1. Two libraries derived from qualitative differential screening aregenerated from mRNA extracts of differentiated PC12 cells in the processof apoptosis following NGF removal on the one hand and fromdifferentiated PC12 cells prevented from undergoing apoptosis bysupplementing IGF-1 on the other hand. To these libraries, may behybridized probes prepared from mRNA derived from differentiated PC12 inthe process of apoptosis and whose survival is enhanced by treatmentwith a neuroprotective product to be tested. The efficiency of the testcompound to reverse the qualitative characteristics can thus beappreciated by monitoring the capacity of the probe to selectivelyhybridize to those specific library clones representing cells having abetter survival rate. This test could be subsequently used to test theefficiency of derivatives of such a compound or any other novel familyof neuroprotective compounds and to improve the pharmacological profilethereof.

In a specific embodiment, the method of the invention allows one toassess the efficacy of a neuroprotective test compound by carrying outhybridization with a differential library according to the inventionderived from a healthy nerve cell and this neurodegenerative model cell.

In another embodiment, one is interested in testing an antitumorcompound using differential libraries established from tumor and healthycell samples.

As already noted, the method of the invention could furthermore be usedto improve the properties of a compound, by testing the capacity ofvarious derivatives thereof to induce a hybridization profile similar tothat of the library representative of the healthy sample.

The invention is further directed to the use of the methods, nucleicacids or libraries described hereinabove in pharmacogenomics, i.e. toassess (predict) the response of a patient to a test compound ortreatment.

Pharmacogenomics is aimed at establishing genetic profiles of patientswith a view to determine which treatment would reasonably be successfulfor a given pathology. The techniques described in the present inventionmake it possible in this respect to establish cDNA libraries that arerepresentative of qualitative differences occurring between apathological condition which is responsive to a given treatment andanother condition which is unresponsive or poorly responsive thereto,and thus may qualify for a different therapeutic strategy. Once thesestandard libraries are established, they can be hybridized with probesprepared from the patients messenger RNAs. The hybridization resultsallow one to determine which patient has a hybridization profilecorresponding to the responsive or non responsive condition and thusrefine treatment choice in patient management.

In this application, the purpose is on the one hand to suggest dependingon the patient's history the most appropriate treatment regimen likelyto be successful and on the other hand to enroll in a given treatmentregimen those patients most likely to benefit therefrom. As with otherapplications, two qualitative differential screening libraries areprepared: one based on a pathological model or sample known to respondto a given treatment, and another based on a further pathological modelor sample which is poorly responsive or unresponsive to therapy. Thesetwo libraries are then hybridized with probes derived from mRNAsextracted from biopsy tissues of individual patients. Depending onwhether such probes preferentially hybridize with the alternativelyspliced forms specific to one particular condition, the patients may bedivided into responsive and unresponsive subjects to the standardtreatment which initially served to define the models.

In this respect, the invention is also directed to a method fordetermining or assessing the response of a patient to a test compound ortreatment comprising hybridizing:

-   -   differential libraries between cDNAs and RNAs from a biological        sample responsive to said compound/treatment and from a        biological sample which is poorly responsive or unresponsive to        said compound/treatment, with,    -   a nucleic acid preparation derived from a pathological        biological sample of the patient, and    -   assessing the responsiveness of the patient by determining the        extent of hybridization with the different libraries.

A preferred example of the usefulness of qualitative differentialscreening in pharmacogenomics is illustrated by a qualitativedifferential screening between two tumors of the same histologicalorigin, one of which showing regression when treated with an antitumorcompound (for example transfer of cDNA coding for wild type p53 proteinby gene therapy), while the other being unresponsive to such treatment.The first benefit derived from constructing qualitative differentiallibraries between these two conditions is the ability to determine, byanalyzing clones making up these libraries, which molecular mechanismsare elicited during regression as observed in the first model and absentin the second.

Subsequently, the use of filters or any other support material bearingcDNAs derived from these libraries allows one to conduct hybridizationwith probes derived from mRNAs of tumor biopsies whose response to saidtreatment is to be predicted. It is possible by looking at the resultsto assign patients to an optimized treatment regimen.

One particular example of this method consists in determining the tumorresponse to p53 tumor suppressor gene therapy. It has indeed beenreported that certain patients and certain tumors respond more or lessto this type of treatment (Roth et al., (1995) Nature Medicine, 2: 958).It is therefore essential to be able to determine which types of tumorsand/or which patients are sensitive to wild type p53 gene therapy, inorder to optimize treatment and make the best choice regarding theenrollment of patients in clinical trials being undertaken.Advantageously, the method of the invention makes it possible tosimplify the procedure by providing libraries specific to qualitativecharacteristics of p53responsive cells and non responsive cells.Examples of cell models sensitive or resistant to p53 are described forinstance by Sabbatini et al. (Genes Dev., (1995), 9: 2184) or by Roemeret al. (Oncogene, (1996), 12: 2069). Hybridization of these librarieswith probes derived from patients' biopsy samples will make assessmentof patient responsiveness easier. In addition, the specific librarieswill allow identification of nucleic acids involved in p53responsiveness.

The present application is therefore also directed to the establishmentof differential screening libraries from pathological samples, orpathological models, which vary in responsiveness to at least onepharmacological agent. These libraries can be restricted, complex orautologous libraries as defined supra. It is also concerned with thespreading of these libraries upon filters or support materials known tothose skilled in the art (nitrocellulose, nylon . . . ). In anadvantageous manner, these support materials may be chips which thusdefine pharmacogenomic chips. The invention further relates to thepotential exploitation of sequencing data of different clones formingsuch libraries with a view to elucidate the mechanisms which lead thepathological samples to respond differently to various treatments, aswell as to the use of such libraries for conducting hybridization withprobes derived from biopsy tissue originating from pathologicalconditions one wishes to predict the response to the standard treatmentinitially used to define those libraries.

The present invention thus describes that variations in splicing formsand/or patterns represent sources of pharmacogenomic markers, i.e.sources of markers by which to determine the capacity of and the mannerin which a patient will respond to treatments. In this respect, theinvention is thus further directed to the use of inter-individualvariability in the isoforms generated by alternative splicing(spliceosome analysis) as a source of pharmacogenomic markers. Theinvention also concerns the use of splicing modifications induced bytreatments as a source of pharmacogenomic markers. Thus, as explainedhereinabove, the DATAS methods of the invention make it possible togenerate nucleic acids representative of qualitative differencesoccurring between two biological samples. Such nucleic acids, orderivatives thereof (probes, primers, complementary acids, etc.) may beused to analyze the spliceosome of subjects, with a view todemonstrating their capacity and manner of responding to treatments, ortheir predisposition to a given treatment pathology, etc.

These various general examples illustrate the usefulness of qualitativedifferential screening libraries in studies of genotoxicity,genopharmacology and pharmacogenomics as well as in research onpotential diagnostic or therapeutic targets. Such libraries are derivedfrom cloning the qualitative differences occurring between twopathophysiological situations. Since another use of the cDNAsrepresentative of these qualitative differences is to generate probesdesigned to screen a genomic DNA library whose characteristics aredescribed hereinabove, such an approach may also be implemented for anystudy of genotoxicity, genopharmacology and pharmacogenomics as well asfor gene identification. In genotoxicity studies for instance, genomicclones statistically restricted by the size of their insertions to asingle intron or to a single exon are arranged on filters according totheir hybridization with DATAS probes derived from qualitativedifferential analysis between a reference cell or tissue sample and thesame cells or tissues treated by a reference toxic compound. Once suchclones representative of different classes of toxicity are selected,they can then be hybridized with a probe derived from total messengerRNAs of a same cell population or a same tissue sample treated by acompound whose toxicity is to be assessed.

Other advantages and practical applications of the present inventionwill become more apparent from the following examples which are givenfor purposes of illustration and not by way of limitation. The fields ofapplication of the invention are shown in FIG. 7.

LEGENDS TO FIGURES

FIG. 1. Schematic representation of differential screening assaysaccording to the invention (FIG. 1A) using one (FIG. 1B) or two (FIG.1C) hybridization procedures, and use of nucleic acids (FIG. 1D).

FIG. 2. Schematic representation of the production of RNA/DNA hybridsallowing characterization of single stranded RNA sequences, specificmarkers of the pathological or healthy state.

FIG. 3. Schematic representation of a method for isolating andcharacterizing by sequencing single stranded RNA sequences specific to apathological or healthy condition.

FIG. 4. Schematic representation of another means for characterizing bysequencing all or part of the single stranded RNAs specific to apathological or healthy condition.

FIG. 5. Schematic representation of the isolation of alternativelyspliced products based on R-loop structures.

FIG. 6. Schematic representation of qualitative differential screeningby loop restriction (formation of ds cDNA/cDNA homoduplexes andextraction of data, FIG. 6A) and description of the data obtained (FIG.6B).

FIG. 7. Benefits of qualitative differential screening at differentstages of pharmaceutical research and development.

FIG. 8. Isolation of a differentially spliced domain in the grb2/grb33model. A) Production of synthetic grb2 and grb33 RNAs. B) Description ofthe first steps of DATAS leading to characterization of an RNA fragmentcorresponding to a differentially spliced domain; 1: grb2 RNA, 2:Hybridization between grb2 RNA and grb33 cDNA, 3: Hybridization betweengrb2 RNA and grb2 cDNA, 4: Hybridization between grb2 RNA and water, 5:Supernatant after passage of (2) on streptavidin beads, 6: Supematantafter passage of (3) on streptavidin beads, 7: Supernatant after passageof (4) on streptavidin beads, 8: RNase H digestion of grb2 RNA/grb33cDNA duplex, 9: RNase H digestion of grb2 RNA/grb2 cDNA duplex, 10:RNase H digestion of grb2 RNA, 11: same as (8) after passage on anexclusion column, 12: same as (9) after passage on an exclusion column,13: same as (10) after passage on an exclusion column.

FIG. 9. Representation of unpaired RNAs derived from RNase H digestionof RNA/single stranded cDNA duplexes originating from HepG2 cellstreated or not by ethanol.

FIG. 10. Representation of double stranded cDNAs generated by one of theDATAS variants. 1 to 12: PCR on RNA loop populations derived from RNaseH digestion, 13: PCR on total cDNA.

FIG. 11. Application of the DATAS variant involving double stranded cDNAin the grb2/grb33 model. A) Agarose gel analysis of the complexesfollowing hybridization: 1: double stranded grb2 cDNA/grb33 RNA, 2:double stranded grb2 cDNA/grb2 RNA, 3: double stranded grb2 cDNA/water.B) Digestion of samples 1, 2 and 3 in (A) by nuclease S1 and mung beannuclease: 1 to 3: complexes 1 to 3 before glyoxal treatment; 4 to 6:complexes 1 to 3 after glyoxal treatment; 7 to 9: Nuclease S1 digestionof 1 to 3; 10 to 12: Mung bean nucdease digestion of 1 to 3.

FIG. 12. Application of the DATAS variant involving single stranded cDNAand RNase H in a HepG2 cell system treated or not with 0.1 M ethanol for18 hours. Cloned inserts were transferred to a membrane after agarosegel electrophoresis and hybridized with probes corresponding to thetreated (Tr) and untreated (NT) conditions.

FIG. 13. Experimental procedure for assessing the toxicity of a product.

FIG. 14. Experimental procedure for monitoring the efficacy of aproduct.

FIG. 15. Experimental procedure for investigating the sensitivity of apathological condition to a treatment.

FIG. 16. Analysis of differential hybridization of clones derived fromDATAS using RNAs from induced cells and cDNAs from non-induced cells. A)Use of bacterial colonies deposited and lysed on a membrane. B) Southernblot on a selection of dones from A.

FIG. 17. Nucleotide and peptide sequence of ΔSHC (SEQ ID NO: 9 and 10).

FIG. 18. Cytotoxicity and apoptosis tests on HepG2 cells treated with A)ethanol; B) camptothecin; C) PMA.

FIG. 19. RT-PCR reactions using RNAs derived from HepG2 cells treated ornot (NT) with ethanol (Eth.), camptothecin (Camp.) and PMA (PMA)allowing amplification of the fragments corresponding to MACH-a, BCL-X,FASR domains and using beta-actin as normalization control.

In the examples and the description of the invention, reference is madeto sequences from the List of Sequences, which contains the followingfree text:

-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO-   <223>OLIGO

EXAMPLES

1. Differential Cloning of Alternative Splicing and Other QualitativeModifications in RNAs Using Single Stranded cDNAs

Messenger RNAs corresponding to two conditions, one being normal (mN)and the other being of a pathological origin (mP), are isolated frombiopsy samples or cultured cells. These messenger RNAs are convertedinto complementary DNAs (cN) and (cP) by means of reverse transcriptase(RT). mN/cP and cN/mP hybrids are then prepared in a liquid phase (seethe diagram of FIG. 2 illustrating one of either cases leading to theformation of cN/mP).

These hybrids are advantageously prepared in phenol emulsion (PERTtechnique or Phenol Emulsion DNA Reassociation Technique) continuouslysubjected to thermocycling (Miller, R., D. and Riblet, R., (1995),Nucleic Acids Research, 23 (12): 2339-2340). Typically, thishybridization is executed using between 0.1 and 1 μg of polyA+ RNA and0.1 to 2 μg of complementary DNA in an emulsion formed of an aqueousphase (120 mM sodium phosphate buffer, 2.5 M NaCl, 10 mM EDTA) and anorganic phase representing 8% of the aqueous phase and formed of twicedistilled phenol.

Another method is also advantageously employed to obtain theheteroduplexes: after the reverse transcription reaction, the newlysynthesized cDNA is separated from the biotinylated oligodT primer byexclusion chromatography. 0.1 to 2 μg of this cDNA is coprecipitatedwith 0.1 to 1 μg of polyA+ RNA in the presence of 0.3 M sodium acetateand two volumes of ethanol. These coprecipitated nucleic acids are takenup in 30 μl of a hybridization buffer composed of 80% formamide, 40 mMPIPES (piperazinebis(2-ethanesulfonic acid)) pH 6.4, 0.4 M NaCl and 1 mMEDTA.

The nucleic acids in solution are heat-denatured at 85° C. for 10 minand hybridization is then carried out at 40° C. for at least 16 h and upto 48 h.

The advantage of the formamide hybridization procedure is that itprovides more highly selective conditions for cDNA and RNA strandpairing.

As a result of these two hybridization techniques there is obtained anRNA/DNA heteroduplex the base pairing extent of which depends on theability of RT to synthesize the entire cDNA. Other single strandedstructures observed are RNA (and DNA) regions corresponding toalternative splicings which distinguish the two pathophysiologicalstates under study.

The method is then aimed at characterizing the genetic information borneby such splice loops.

To this end, the heteroduplexes are purified by capture of cDNAs (primedwith biotinylated oligo-dT) by means of streptavidin-coated beads.Advantageously these beads are beads having magnetic properties,allowing them to be separated from RNAs not engaged in the heteroduptexstructures by the action of a magnetic separator. Such beads and suchseparators are commercially available.

At this stage of the procedure are isolated heteroduplexes and cDNAs notengaged in hybridization with RNAs. This material is then subjected tothe action of RNase H which will selectively hydrolyze regions of RNAhybridized with cDNAs. The products of this hydrolysis are on the onehand cDNAs and on the other hand, RNA fragments which correspond tosplice loops or non hybridized regions as a result of incomplete reversetranscriptase reaction. The RNA fragments are separated from DNA bymagnetic separation according to the same experimental procedure as setforth above and by digestion with DNase free of contaminating RNaseactivity.

1.1. Validation of the DATAS Method on Splicing Variants of the Grb2Gene

The feasibility of this approach was demonstrated in an in vitro systemusing RNA corresponding to the coding region of Grb2 on the one hand andsingle stranded cDNA complementary to the coding region of Grb3.3. TheGrb2 gene has an open reading frame of 651 base pairs. Grb33 is anisoform of grb2 generated by alternative splicing and comprising adeletion of 121 base pairs in the SH2 functional domain of grb2 (Fath etal., (1994), Science 264: 971-4). Grb2 and Grb33 RNAs are synthesized bymethods known to those skilled in the art from a plasmid harboring theGrb2 or Grb33 coding sequence driven by the T7 promoter by means of theRiboMax kit (Promega). Analysis of the products shows that the synthesisis homogeneous (FIG. 8A). For purposes of visualization, Grb2 RNA wasalso radiolabeled by incorporation of a labeled base during in vitrotranscription by means of the RiboProbe kit (Promega). Grb2 and Grb33cDNAs were synthesized by reverse transcription from the above-obtainedsynthetic RNA products, using the Superscript II kit (Life Technologies)and a biotinylated oligonucleotide primer common to Grb2 and Grb33corresponding to the complement of the Grb2 sequence (618-639). RNAs andcDNAs were treated according to the suppliers' instructions (Promega,Life Technologies), purified on an exclusion column (RNase-free SephadexG25 or G50, 5 Prime, 3 Prime) and quantified by spectrophotometry.

The first steps of DATAS were executed by combining in suspension 10 ngof labeled Grb2 RNA with:

-   -   1. 100 ng of biotinylated grb33 cDNA,    -   2. 100 ng of biotinylated grb2 cDNA,    -   3. water        in 30 μl of a hybridization buffer containing 80% formamide, 40        mM PIPES (pH 6.4), 0.4 M NaCl, 1 mM EDTA. The nucleic acids are        denatured by heating for 10 min at 85° C., after which the        hybridization is carried out for 16 hours at 40° C. After        capture on streptavidin beads, the samples are treated with        RNase H as described hereinabove.

These steps are analyzed by electrophoresis on a 6% acrylamide gelfollowed by processing of the gels with an Instant Imager (PackardInstruments) which allows the qualification and quantification of thespecies derived from labeled grb2 RNA (FIG. 8B). Thus, lanes 2, 3 and 4show that grb2/grb33 and grb2/grb2 duplexes are formed quantitatively.Migration of the grb2/grb33 complex is slower relative to that of grb2RNA (lane 2) while that of the grb2/grb2 complex is faster (lane 3).Lanes 5, 6 and 7 correspond to samples not retained by the streptavidinbeads showing that 80% of grb2/grb33 and grb2/grb2 complexes werecaptured by the beads whereas non-biotinylated grb2 RNA alone was foundsolely in the bead supernatant. Treatment with RNase H releases, inaddition to free nucleotides which migrate faster than bromophenol blue(BPB), a species that migrates below xylene cyanol blue (XC) (indicatedby an arrow in the figure) and this, specifically in lane 8corresponding to the grb2/grb33 complex relative to lanes 9 and 10 whichcorrespond to the grb2/grb2 complex and to grb2 RNA. Lanes 11, 12 and 13correspond to lanes 8, 9 and 10 after passage of the samples through anexclusion column to remove free nucleotides. The migration observed inlanes 8 and 11 is that expected for an RNA molecule corresponding to the121-nucleotide deletion that distinguishes grb2 from grb33.

This result clearly shows that it is possible to obtain RNA loopsgenerated by the formation of heteroduplex between two sequences derivedfrom two splicing isoforms.

1.2. Application of the DATAS Method to Generate Qualitative Librariesof Hepatic Cells in a Healthy and Toxic State

A more complex situation was examined. Within the scope of theapplication of DATAS technology as a tool to predict the toxicity ofmolecules, the human hepatocyte cell line HepG2 was treated with 0.1 Methanol for 18 hours. RNAs were extracted from cells that were or werenot subjected to treatment. The aforementioned DATAS variant(preparation of biotinylated ss cDNA, cross hybridizations in liquidphase, application of a magnetic field to separate the species, RNase Hdigestion) was effected with untreated cells in the reference condition(or condition A) and with treated cells in the test condition (orcondition B) (FIG. 9). As the extracted RNAs were not radiolabeled, theRNAs generated by RNase H digestion were visualized by carrying out anexchange reaction to replace the RNA 5′ phosphate with a labeledphosphate, by means of T4 polynucleotide kinase and gamma-P³²ATP. Theselabeled products were then loaded on an acrylamide/urea gel and analyzedby exposure using an Instant Imager (Packard Instruments). Complexsignatures derived from A/B and B/A hybridizations could then bevisualized with a first group of signals migrating slowly in the gel andcorresponding to large nucleic acid sequences and a second group ofsignals migrating between 25 and 500 nucleotides. These signatures areof much lower intensity in condition A/A. suggesting that ethanol caninduce a reprogramming of RNA splicing events, manifested as thepresence of A/B and B/A signals.

1.3. Cloning and Preparation of Libraries from the Identified NucleicAcids

Several experimental alternatives may then be considered to clone theseRNA fragments resistant to the action of RNase H:

A. A first approach consists in isolating and cloning such loops (FIG.3).

According to this approach, one proceeds with ligation ofoligonucleotides to each end by means of RNA ligase according toconditions known in the art. These oligonucleotides are then used asprimers to effect RT PCR. The PCR products are cloned and screened withtotal complementary DNA probes corresponding to the twopathophysiological conditions of interest. Only those clonespreferentially hybridizing with one of either probes contain the spliceloops which are then sequenced and/or used to generate libraries.

B. The second approach (FIG. 4) consists in carrying out a reversetranscription reaction on single stranded RNA released from theheteroduplex structures by RNase H digestion, initiated by means of atleast partly random primers. Thus, these may be primers with random 3′and 5′ sequences, primers with random 3′ ends and defined 5′ sequences,or yet semi-random oligonucleotides, i.e. comprising a region ofdegeneration and a defined region.

According to this strategy, the primers may therefore hybridize eitheranywhere along the single stranded RNA, or at each succession of basesdetermined by the choice of semi-random primer. PCR is then run usingprimers corresponding to the above-described oligonucleotides in orderto obtain splice loop-derived sequences.

FIG. 10 (lanes 1 to 12) presents the acrylamide gel analysis of the PCRfragments obtained in several DATAS experiments and coupled to the useof the following semi-random oligonucleotides:GAGAAGCGTTATNNNNNNNAGGT  (SEQ ID NO: 1, X=T)GAGAAGCGTTATNNNNNNNAGGA  (SEQ ID NO: 1, X=A)GAGAAGCGTTATNNNNNNNAGGC  (SEQ ID NO: 1, X=C)GAGAAGCGTTATNNNNNNNAGGG  (SEQ ID NO: 1, X=G)

Comparing these results with the complexity of the signals obtainedusing the same oligonucleotides, but with total cDNA as the template(lane 13), demonstrates that DATAS makes it possible to filter (profile)the information corresponding to qualitative differences.

This variant was used to clone an event corresponding to the grb2 RNAdomain generated by RNase H digestion of the grb2 RNA/grb33 singlestranded cDNA duplex according to the above-described protocol (example1.1). To do so, an oligonucleotide with the sequence:GAGAAGCGTTATNNNNNNNNTCCC (SEQ ID NO: 2), chosen from the modelGAGAAGCGTTATNNNNNNNWXYZ (where N is defined as above, W, X and Y eachrepresent a defined fixed base, and Z designates either a defined base,or a 3′-OH group, SEQ ID NO: 3) and selected so as to amplify a fragmentin the grb2 deletion, was used, allowing generation of a PCR fragmentwhich, after cloning and sequencing, was shown to indeed be derived fromthe grb2 deleted domain (194-281 in grb2).

These two approaches therefore allow the production of nucleic acidcompositions representative of the differential splicings in bothconditions being tested, which may be used as probes or to constructqualitative differential cDNA libraries. The capacity of DATAStechnology to generated profiled cDNA libraries representative ofqualitative differences is further illustrated in example 1.4 below.

1.4. Production of Profiled Libraries Representative of HumanEndothelial Cells

This example was carried out using a human endothelial cell line(ECV304). The qualitative analysis of gene expression was achieved byusing cystolic RNA extracted from growing cells, on the one hand, andfrom cells in the process of anoikis (apoptosis induced by removing theadhesion support), on the other hand.

ECV cells were grown in 199 medium supplemented with Earle salts (LifeSciences). Anoikis was induced by passage for 4 hours onpolyHEMA-treated culture dishes. For RNA preparation, cells were lysedin a buffer containing Nonidet P-40. Nuclei are then eliminated bycentrifugation. The cytoplasmic solution was then adjusted so as tospecifically fix the RNA to the Rneasy silica matrix according to theinstructions of the Quiagen company. After washing, total RNA is elutedin DEPC-treated water. Messenger RNAs are prepared from total RNAs byseparation on Dynabeads oligo (dT)₂₅ magnetic beads (Dynal). Aftersuspending the beads in a fixation buffer, total RNA is incubated for 5min at room temperature. After magnetic separation and washing, thebeads are taken up in elution buffer and incubated at 65° C. to releasemessenger RNAs.

The first DNA strand is synthesized from the messenger RNA by means ofSuperScript II or ThermoScript reverse transcriptase (Life Technologies)and olido-(dT) primers. After RNase H digestion, free nucdeotides areeliminated by passage through a Sephadex G50 (5 Prime-3 Prime) column.Following phenol/chloroform extraction and ethanol precipitation,samples are quantified by UV absorbance.

The required quantifies of RNA and cDNA (in this case 200 ng of each)are pooled and ethanol-precipitated. The samples are taken up in avolume of 30 μl in hybridization buffer (40 mM Hepes (pH 7.2), 400 mMNaCl, 1 mM EDTA) supplemented with deionized formamide (80% (v/v),except if otherwise indicated). After denaturation for 5 min at 70° C.,samples are incubated overnight at 40° C.

The streptavidin beads (Dynal) are washed then reconditioned in fixationbuffer (2×=10 mM Tris-HCl (pH 7.5), 2 M NaCl, 1 mM EDTA). Thehybridization samples are diluted to a volume of 200 μl with water, thenadjusted to 200 μl of beads and incubated for 60 min at 30° C. Aftermagnetic capture and washing of the beads, the latter are suspended in150 μl of RNase H buffer then incubated for 20 min at 37° C. Aftermagnetic capture, nonhybridized regions are released into thesupernatant which is treated with Dnase, then extracted with acidicphenol/chloroform and ethanol-precipitated. Ethanol precipitations ofsmall quantities of nucleic acids are carried out using a commercialpolymer SeeDNA (Amersham Pharmacia Biotech) allowing quantitativerecovery of nucleic acids from very dilute solutions (in the ng/mlrange).

Synthesis of cDNA from the RNA samples derived from RNase H digestion iscarried out by means of random hexanucleotides and Superscript IIreverse transcriptase. The RNA is then digested with a mixture of RNaseH and RNase T1. The primer, the unincorporated nucleotides and theenzymes are separated from the cDNA by means of a GlassMAX Spincartridge. The cDNA corresponding to splice loops is then subjected toPCR using the semi-random oligonucleotides described hereinabove in theinvention. In this case the chosen oligonucleotides are as follows:GAGAAGCGTTATNNNNNCCA  (SEQ ID NO: 4)

The PCR reaction is effected using Taq Polymerase for 30 cycles:

-   -   Initial denaturation: 94° C. for 1 min.    -   94° C. for 30 s    -   55° C. for 30 s    -   72° C. for 30 s    -   Final elongation: 72° C. for 5 min.

The PCR products are cloned into the pGEM-T vector (Promega) with afloating T at the 3′ ends so as to simplify cloning of the fragmentsderived from the activity of Taq polymerase. After transformation incompetent JM109 bacterial (Promega), the resulting colonies aretransferred to nitrocellulose filters, and hybridized with probesderived from the products of PCR carried out on total cDNA from growingcells on the one hand and in anoikis on the other hand. The sameoligonucleotides GAGAAGCGTTATNNNNNCCA (SEQ ID NO: 4) are used for thesePCR reactions. In a first experimental embodiment, 34 clonespreferentially hybridizing with the probe from cells in apoptosis and 13clones preferentially hybridizing with the probe from growing cells werelocated.

Among these 13 clones, 3 clones contain the same cDNA fragment derivedfrom the SH2 domain of the SHC protein.

This fragment has the following sequence:CCACACCTGGCCAGTATGTGCTCACTGGCTTGCAGAGTGGGCAGCCAGCCTAAGCATTTGCACTGG  (SEQID NO: 5)

The use of PCR primers flanking the SHC SH2 domain (5′ oligo:GGGACCTGTTTGACATGAAGCCC (SEQ ID NO:6); 3′ oligo: CAGTCCGCTCCACAGGTTGC(SEQ ID NO:7)) allowed characterization of the SHC SH2 domain deletionwhich is specifically observed in ECV cells in anoikis. With this primerpair, a single amplification product corresponding to a 382 base paircDNA fragment which contains the intact SH2 domain is obtained from RNAfrom exponentially growing ECV cells. A further 287 base pair fragmentis observed when the PCR is carried out with RNA from cells in anoikis.This additional fragment derives from a messenger RNA derived from theSCH messenger but with a deletion.

This deletion has the following sequence:GTACGGGAGAGCACGACCACACCTGGCCAGTATGTGCTCACTGGCTTGCAGAGTGGGCAGCCTAAGCATTTGCTACTGGTGGACCCTGAGGGTGTG  (SEQID NO: 8).

This deletion corresponds to bases 1198 to 1293 of the messenger openreading frame encoding the 52 kDa and 46 kDa forms of the SHC protein(Pelicci, G. et al., (1992), Cell, 70: 93-104).

Structural data on the SH2 domains together with the literature indicatethat such a deletion leads to the loss of affinity for phosphotyrosinessince it encompasses the amino acids involved in interactions withphosphorylated tyrosines (Waksman, G. et al., (1992), Nature, 358:646-653). As SHC proteins are adaptors which link different partners viatheir SH2 and PTB domains (PhosphoTyrosine Binding domain), thisdeletion therefore generates a native negative dominant form of SHCwhich we call ΔSHC As the SH2 domains of proteins for which the geneshave been sequenced are carried on two exons, it is likely that thedeletion identified by DATAS corresponds to an alternative exon of theSHC gene.

The protein and nucleic acid sequences of ΔSHC are given in FIG. 17 (SEQID NO: 9 and 10).

As the SHC SH2 domain is involved in the transduction of numeroussignals involved in cell proliferation and viability, examination of theΔSHC sequence makes it possible to predict its negative dominantproperties on the SHC protein and its capacity to interfere with variouscellular signals.

The invention equally concerns this new spliced form of SHC, the proteindomain corresponding to the splicing, any antibody or nucleic acid probeallowing its detection in a biological sample, and their use fordiagnostic or therapeutic purposes, for example.

The invention particularly concerns any SHC variant comprising at leastone deletion corresponding to bases 1198 to 1293, more particularly adeletion of sequence SEQ ID NO: 8. The invention more specificallyconcerns the ΔSHC variant possessing the sequence SEQ ID NO: 9, coded bythe sequence SEQ ID NO: 10.

The invention therefore concerns any nucleic acid probe, oligonucleotideor antibody by which to identify the hereinabove ΔSHC variant, and/orany alteration of the SHC/ΔSHC ratio in a biological sample. This maynotably be a probe or oligonucleotide complementary to all or part ofthe sequence SEQ ID NO: 8, or an antibody directed against the proteindomain encoded by this sequence. Such probes, oligonudeotides orantibodies make it possible to detect the presence of the nonsplicedform (eg., SHC) in a biological sample.

The materials may further be used in parallel with the probes,oligonucleotides and/or antibodies specific of the spliced form (eg.,ΔSHC), i.e. corresponding for example to the junction region resultingfrom splicing (located around nucaeotide 1198 in sequence SEQ ID NO:10).

Such materials may be used for the diagnosis of diseases related toimmune suppression (cancer, immunosuppressive therapy, AIDS, etc.).

The invention also concerns any screening method for molecules based onblocking (i) the spliced domain in the SHC protein (especially in orderto induce a state of immune tolerance for example in autoimmune diseasesor graft rejection and cancer) or (ii) the added functions acquired bythe ΔSHC protein.

The invention is further directed to the therapeutic use of ΔSHC, andnotably to the treatment of cancerous cells or cancers (ex vivo or invivo) in which SHC protein hyperphosphorylation can be demonstrated, forexample. In this respect, the invention therefore concerns any vector.notably a viral vector, comprising a sequence coding for ΔSHC. Thisvector is preferably capable of transfecting cancerous or growing cells,such as smooth muscle cells, endothelial cells (restenosis), fibroblasts(fibrosis), preferably of mammalian, notably human, origin. Viralvectors may be exemplified in particular by adenoviral, retroviral, AAV,herpes vectors, etc.

2. Differential Cloning of Alternative Splicing and Other QualitativeModifications of RNA Using Double Stranded cDNA (FIG. 5)

Messenger RNAs corresponding to normal (mN) and pathological (mP)conditions are produced, as well as corresponding double strandedcomplementary DNAs (dsN and dsP) by standard molecular biologyprocedures. R-loop structures are then obtained by hybridizing mN withdsP and mP with dsN in a solution containing 70% formamide.Differentially spliced nucleic acid domains between conditions N and Pwill remain in the form of double stranded DNA. Displaced singlestranded DNAs are then treated with glyoxal to avoid furtherdisplacement of the RNA strand upon removal of formamide. After removalof formamide and glyoxal and treatment with RNase H, there are obtainedbee-type structures, the unpaired single stranded DNAs beingrepresentative of the bee wings and the paired double stranded domain ofinterest being reminiscent of the bee's body. The use of enzymes whichspecifically digest single stranded DNA such as nuclease Si or mung beannuclease allows the isolation of DNA that has remained in doublestranded form, which is next cloned and sequenced. This second techniqueallows for direct formation of a double stranded DNA fingerprint of thedomain of interest, when compared to the first procedure which yields anRNA fingerprint of this domain.

This approach was carried out on the grb2/grb33 model described above.Grb2 double stranded DNA was produced by PCR amplification of grb2single stranded cDNA using two nucleotide primers corresponding to thesequence (1-22) of grb2 and to the complementary sequence (618-639) ofgrb2. This PCR fragment was purified on an agarose gel, cleaned on anaffinity column (JetQuick, Genomed) and quantified by spectrophotometry.At the same time, two synthetic RNAs corresponding to the grb2 and grb33reading frames were produced from plasmid vectors harboring grb2 orgrb33 cDNAs under the control of the T7 promoter, by means of theRiboMax kit (Promega). The RNAs were purified as instructed by thesupplier and cleaned on an exclusion column (Sephadex G50, 5 prime-3prime). 600 ng of double stranded grb2 DNA (1-639) were combined with:

-   -   1. 3 μg of grb33 RNA    -   2. 3 μg of grb2 RNA    -   3. water        in three separate reactions, in the following buffer:

100 mM PIPES (pH 7.2), 35 mM NaCl, 10 mM EDTA, 70% deionized formamide(Sigma).

The samples were heated to 56° C., then cooled to 44° C. by −0.2° C.increments every 10 minutes. They are then stored at 4° C. Analysis ofthe agarose gel reveals the altered migration patterns of lanes 1 and 2as compared with the control lane 3 (FIG. 11A), indicating that newcomplexes were formed. Samples are then treated with deionized glyoxal(Sigma) (5% v/v or 1 M) for 2 h at 12° C. The complexes are thenprecipitated with ethanol (0.1 M NaCl, 2 volumes of ethanol), washedwith 70% ethanol, dried, then resuspended in water. They are nexttreated by RNase H (Life Technologies), then by an enzyme specific forsingle stranded DNA. Nuclease S1 and mung bean nuclease have such aproperty and are commercially available (Life Technologies, Amersham).Such digestions (incubations for 5 minutes in the buffers supplied withthe enzymes) were analyzed on agarose gels (FIG. 11B). Significantdigest products were obtained only from the complexes derived fromreaction 1 (grb2/grb33) (FIG. 11B, lanes 7 and 10). These digestionsappear more complete with nuclease S1 (lane 7) than with mung beannuclease (lane 10). Thus, the band corresponding to a size slightlygreater than 100 base pairs (indicated by an arrow on lane 7) waspurified, cloned into the pMos-Blue vector (Amersham) and sequenced.This fragment corresponds to the 120 base pair domain of grb2 which isdeleted in grb33.

This approach may now be implemented starting with a total messenger RNApopulation and a total double stranded cDNA population producedaccording to methods known to those skilled in the art. RNAscorresponding to the reference condition are hybridized with doublestranded cDNAs derived from the test condition and vice versa. Afterapplication of the hereinabove protocol, the digests are loaded onagarose gels so as to isolate and purify the bands corresponding tosizes ranging from 50 to 300 base pairs. Such bands are then cloned in avector (pMos-Blue, Amersham) to generate a library of inserts enrichedin qualitative differential events.

3. Construction of Libraries Derived from Qualitative DifferentialScreening

The two examples described hereinabove lead to the cloning of cDNAsrepresentative of all or part of differentially spliced sequencesoccurring between two given pathophysiological conditions. These cDNAsallow the construction of libraries by insertion of such cDNAs intoplasmid or phage vectors. These libraries may be deposited onnitrocellulose filters or any other support material known in the art,such as chips or biochips or membranes. The aforementioned libraries maybe stored in a cold place, away from light. These libraries, oncedeposited and fixed on support materials by conventional techniques, maybe treated by compounds to eliminate the host bacteria which allowed thereplication of the plasmids or phages. These libraries may also beadvantageously composed of cDNA fragments corresponding to cloned cDNAsbut prepared by PCR so as to deposit on the filter only those sequencesderived from alternative splicing events.

One of the features as well as one of the original characteristics ofqualitative differential screening is that this method advantageouslyleads to not only one but two differential libraries (“library pair”)which represent the whole array of qualitative differences occurringbetween two given conditions. In particular, one of the differentialsplicing libraries of the invention represents the unique qualitativemarkers of the test physiological condition as compared to the referencephysiological condition, while the other library represents the uniquequalitative markers of the reference physiological condition in relationto the test physiological condition. This couple of libraries is equallytermed a library pair or “differential splicing library”.

As one of the benefits of qualitative differential screening is that itmakes it possible to assess the toxicity of a compound, as will be setforth in the next section, a good example of the implementation of thetechnology is the use of DATAS to obtain cDNA dones corresponding tosequences specific of untreated HepG2 cells, on the one hand, andethanol-treated cells, on the other hand. The latter cells exhibit signsof cytotoxicity and DNA degradation via internucleosomal fragmentationstarting from 18 hours of exposure to 1 M ethanol. In order to obtainearly markers of ethanol toxicity, messenger RNAs were prepared fromuntreated cells and from cells treated with 0.1 M ethanol for 18 h.After execution of the DATAS variant which makes use of single strandedcDNA and RNase H, the resulting cloned cDNAs were amplified by PCR,electrophoresed on agarose gels and then transferred to a nylon filteraccording to techniques known to those skilled in the art. For each setof clones specific on the one hand of specific qualitative differencesof the untreated state and on the other hand of sequences specific ofethanol-treated cells, two identical filter duplicates are prepared.Thus the fingerprints of each set of clones are hybridized on the onehand with a probe specific to untreated cells and on the other hand witha probe specific to cells treated with 0.1 M ethanol for 18 h.

The differential hybridization profile obtained and shown in FIG. 12makes it possible to appreciate the quality of the subtraction affordedby the DATAS technique. Thus the clones derived from hybridization ofmRNA from untreated cells (NT) with cDNA from treated cells (Tr) andwhich should correspond to qualitative differences specific of theuntreated condition, hybridize preferentially with a probe representingthe total messenger RNA population of untreated cells. Conversely,clones derived from products resistant to the action of RNase H onRNA(Tr)/cDNA(NT) heteroduplexes hybridize preferentially with a probederived from total messenger RNAs from treated cells.

The two sets of clones specific on the one hand to the treated conditionand on the other hand to the untreated condition represent an example ofqualitative differential libraries characteristic of two distinct cellstates.

4. Uses and Benefits of Qualitive Differential Libraries

The potential applications of the differential splicing libraries of theinvention are illustrated notably in FIGS. 13 to 15. Thus, theselibraries are useful for:

4.1. Evaluating the Toxicity of a Compound (FIG. 13)

In this example, the reference condition is designated A and the toxiccondition is designated B. Toxicity abacus charts are obtained bytreating condition A in the presence of various concentrations of areference toxic compound, for different periods of time. For differentdots of toxicity abacus charts, qualitative differential libraries areconstructed (library pairs), namely in this example, restrictedlibraries rA/cB and rB/cA. The library pairs are advantageouslydeposited on a support. The support is then hybridized with probesderived from the original biological sample treated with different dosesof test compounds: products X, Y and Z. The hybridization reaction isdeveloped in order to determine the toxicity potential of the testproducts: in this example, product Z is highly toxic and product Y showsan intermediate profile. The feasibility of constructing toxicity abacuscharts is clearly illustrated in the aforementioned example regardingthe construction of qualitative differential screening librariesinvolving ethanol treatment and HepG2 cells.

4.2. Assessing the Potency of a Pharmaceutical Composition (FIG. 14)

In this example, a restricted library pair according to the invention isconstructed starting with a pathological model B and a healthy model A(or a pathological model treated with a reference active product). Thedifferential libraries rA/cB and rB/cA are optionally deposited on asupport. This library pair is fully representative of the differences insplicing which occur between both conditions. This library pair allowsthe efficacy of a test compound to be assessed, i.e. to determine itscapacity to generate a “healthy-like” profile (rA/cB) starting from apathological-type profile (rB/cA). In this example, the library pair ishybridized with probes prepared from conditions A and B either treatedor not by the test compound. The hybridization profile that can beobtained is shown in FIG. 14. The feasibility of this application isidentical to that of the aforementioned construction of qualitativedifferential libraries characteristic of healthy and toxic conditions.The toxic condition is replaced by the pathological condition and oneassesses the capacity of a test compound to produce a probe hybridizingmore or less preferentially with the reference or pathologicalconditions.

4.3. Predicting the Response of a Pathological Sample to a Treatment(FIG. 15)

In this example, a restricted library pair according to the invention isconstructed starting with two pathological models, one of which isresponsive to treatment with a given product (the wild type p53 gene forexample): condition A; while the other being unresponsive: condition B.This library pair (rA/cB ; rB/cA) is deposited on a support.

This library pair is then used to determine the sensitivity of apathological test sample to the same product. For that purpose, thislibrary pair is hybridized with probes derived from patients' biopsytissues one wishes to evaluate the response to the reference treatment.The hybridization profile of a responsive biopsy sample and of anunresponsive biopsy sample is presented in FIG. 15.

4.4 Identification of Ligands for Orphan Receptors

The activation of membrane or nuclear receptors by their ligands canspecifically induce regulation defects in the splicing of certain RNAs.Identification of these events by the DATAS methods of the inventionprovides a tool (markers, libraries, kits, etc.) by which to monitorreceptor activation, which can be used to search for natural orsynthetic ligands for receptors, especially orphan receptors. Accordingto this application, markers associated with regulation defects areidentified and deposited on supports. Total cellular RNA,(over)expressing the receptor under study, treated or not by differentcompositions and/or test compounds, is extracted and used as probe in ahybridization reaction with the supports. Detection of hybridizationwith some or even all of the markers deposited on the support, indicatesthat the receptor of interest was activated, and therefore that thecorresponding composition/compound constitutes or contains the ligand ofsaid receptor.

4.5 Identification of Targets of Therapeutic Interest

This is accomplished by identifying genes the splicing of which isaltered in a pathology or in a pathological model and more specificallyby identifying the modified exons or introns. This approach should makeit possible to determine the sequences which code for functional domainsthat are altered in pathologies or in any pathophysiological processinvolving the phenomena of growth, differentiation or apoptosis forexample.

An example of the benefit of qualitative differential screening foridentifying differentially spliced genes is provided by the applicationof DATAS to a model of apoptosis induction via induction of wild typep53 expression. This cellular model was established by transfecting aninducible p53 tumor suppressor gene expression system. In order toidentify qualitative differences which are specifically associated withp53-induced apoptosis, DATAS was implemented starting with messengerRNAs derived from induced and non-induced cells. For these experiments200 ng of polyA+ RNA and 200 ng of cDNA were used for heteroduplexformation. About 100 clones were obtained from each cross hybridization.Hybridization of these bacterial clones, then of the cDNA fragments theycontain, with probes representative of total messenger RNAs from theoriginal conditions allowed identification of sequences specificallyexpressed during the potent p53 induction which leads to cell death(FIG. 16).

These fragments derive from exon or intron sequences which modulate thequality of the message present and qualify the functional domains inwhich they participate or which they interrupt, as targets for treatmentto induce or to inhibit cell death.

Such an approach equally leads to the construction of a library paircomprising all the differential splicing events between a non-apoptoticcondition and an apoptotic condition. This library pair may be used totest the hybridizing capacity of a probe derived from anotherpathophysiological condition or a given treatment. The results of such ahybridization will give an indication as to the potential commitment ofthe gene expression program of the test condition towards apoptosis.

As is apparent from the above description, the invention is furtherconcerned with:

-   -   any nucleic acid probe, any oligonucleotide, any antibody which        recognizes a sequence identified by the method described in the        present application and characterized in that they are        characteristic of a pathological condition,    -   the use of information derived from applying the techniques        disclosed herein for the search of organic molecules for        therapeutic purposes by devising screening assays characterized        in that they target differentially spliced domains occurring        between a healthy and a pathological condition or else        characterized in that they are based on the inhibition of        functions acquired by the protein as a result of differential        splicing,    -   the utilization of the information derived from the methods        described in the present application for gene therapy        applications,    -   the use of cDNAs delivered by gene therapy, wherein said cDNAs        behave as antagonists or agonists of defined cell signal        transduction pathways,    -   any construction or any use of molecular libraries of        alternative exons or introns for purposes of:        -   commercial production of diagnostic means or reagents for            research purposes        -   generation or search of molecules, polypeptides, nucleic            acids for therapeutical applications.    -   any construction or any use of all computerized virtual        libraries containing an array of alternative exons or introns        characterized in that said libraries allow the design of nucleic        acid probes or oligonucleotide primers in order to characterize        alternative splicing forms which distinguish two different        pathophysiological conditions.    -   any pharmaceutical or diagnostic composition comprising        polypeptides, sense or antisense nucleic acids or chemical        compounds capable of interfering with alternative splicing        products identified and cloned by the methods of the invention,    -   any pharmaceutical or diagnostic composition comprising        polypeptides, sense or antisense nucleic acids, or chemical        compounds capable of restoring a splicing pattern representative        of a normal condition in contrast to an alternative splicing        event inherent to a pathological condition.        5. Deregulations of RNA Splicing Mechanisms by Toxic Compounds

This example shows that differential splicing forms and/or profiles maybe used as markers to monitor and/or determine the toxicity and/or theefficacy of compounds.

The effects of toxic compounds on RNA splicing regulation defects weretested as follows. HepG2 hepatocyte cells were treated with differentdoses of three toxic compounds (ethanol, camptothecin, PMA (phorbol12-myristate 13-acetate)). Two cytotoxicity tests (trypan blue, MTT)were performed at different time points: 4 h and 18 h for ethanol; 4 hand 18 h for camptothecin; 18 h and 40 h for PMA.

Trypan blue is a dye that can be incorporated by living cells. Simplecounting of “blue” and “white” cells under a microscope gives thepercentage of living cells after treatment or the percentage ofsurvival. The experimental points are determined in triplicate.

The MTT test is a colorimetric test measuring the capacity of livingcells to convert soluble tetrazolium salts (MTT) into an insolubleformazan precipitate. These dark blue formazan crystals can be dissolvedand their concentration determined by measuring absorbance at 550 nm.Thus, after overnight seeding of 24-well dishes with 150,000 cells,followed by treatment of the cells with the toxic compounds, 50 μl ofMTT (Sigma) are added (at a concentration of 5 mg/ml in PBS). Theformazan crystal formation reaction is carried out for 5 h in a CO2incubator (37° C., 5% CO2, 95% humidity). After addition of 500 μl ofsolubilization solution (0.1 N HCl in isopropanol-Triton X-100 (10%)),the crystals are dissolved with stirring and their absorbance ismeasured at 550 to 660 nm. Determinations are done in triplicate withsuitable controls (viability, cell death, blanks).

A test of apoptosis or programmed cell death was also performed bymeasuring DNA fragmentation with an anti-histone antibody and ELISA. TheCell Death ELISA Plus from Roche was used.

The results of these three tests (FIGS. 18 A, B, C) indicate that thefollowing concentrations:

-   -   ethanol: 0.1 M    -   camptothecin: 1 μg/ml    -   PMA: 50 ng/ml        were well below the measured IC50 values.

HepG2 cells were thus treated with these three concentrations of thesethree compounds for 4 h in the case of ethanol and camptothecin and for18 h in the case of PMA. Messenger RNAs were purified onDynal-Oligo-(dT) beads starting from total RNAs purified with the Rneasykit (Quiagen). cDNA was synthesized from these messenger RNAs usingSuperscript reverse transcriptase (Life Technologies) and randomhexamers as primers

These initial strands served as templates for PCR amplificationreactions (94° C. 1 min, 55° C. 1 min, 72° C. 1 min, 30 cycles) by meansof the following oligonucleotide primers:

MACH-α5′-TGCCCAAATCAACAAGAGC-3′  (SEQ ID NO: 11)5′-CCCCTGACAAGCCTGAATA-3′  (SEQ ID NO: 12)

These primers correspond to the regions common to the differentdescribed isoforms of MACH-α (1, 2 and 3, respectively amplifying 595,550 and 343 base pairs). MACH-α (Caspase-8) is a protease involved inprogrammed cell death (Boldin et al., (1996), Cell, 85: 803-815).

BCL-X5′ ATGTCTCAGAGCAACCGGGAGCTG 3′  (SEQ ID NO: 13)5′ GTGGCTCCATTCACCGCGGGGCTG 3′  (SEQ ID NO: 14)

These primers correspond to the regions common to the differentdescribed isoforms of bcl-X (bcl-Xl, bcl-Xs, BCL-Xβ) (Boise et al.,(1993), Cell 74: 597-608; U72398 (Genbank)) and should amplify a single204 base pair fragment for these three isoforms.

FASR5′-TGCCAAGAAGGGAAGGAGT-3′  (SEQ ID NO: 15)5′-TGTCATGACTCCAGCAATAG3′  (SEQ ID NO: 16)

These primers correspond to the regions common to certain FASR isoformsand should amplify a 478 base pair fragment for wild type form FasR, 452base pairs for isoform Δ8 and 415 for isoform ΔTM.

The results presented in FIG. 19 indicate that:

-   -   Camptothecin induces a decrease in the expression of isoform        MACH-α1 and an increase in isoform MACH-α3.    -   Camptothecin induces the appearance of a new bcl-X isoform        (upper band in the doublet near 200 base pairs).    -   Camptothecin induces a decrease in the wild type form of the fas        receptor, replaced by expression of a shorter isoform which may        correspond to Fas ΔTM.    -   Ethanol induces the disappearance of bcl-x which is replaced by        a shorter isoform.    -   Ethanol induces an increase in the long wild type form of the        fas receptor at the expense of the shorter isoform.

These results demonstrate that treatment with low concentrations oftoxic compounds can induce regulation defects in the alternativesplicings of certain RNAs, and this in a specific manner. Theidentification of these regulation defects at the post-transcriptionallevel, notably by application of DATAS technology, thus constitutes atool to predict the toxicity of molecules.

1. A device for identifying at least one differentially spliced geneproduct, wherein said device comprises a solid support material andsingle-stranded oligonucleotides of between 5 and 100 nucleotides inlength attached to said support material, wherein said oligonucleotidescomprise at least a first and a second oligonucleotide molecule arrangedserially on the support material, wherein said first oligonucleotidemolecule comprises a first sequence that is complementary to andspecific for an exon or an intron of a first gene, and wherein saidfirst sequence corresponds to a region of variability in at least oneproduct of said first gene due to differential splicing, and whereinsaid second oligonucleotide molecule comprises a second sequence that iscomplementary to and specific for an exon-exon or exon-intron junctionregion of said first gene, and wherein said second sequence correspondsto a region of variability in at least one product of said first genedue to differential splicing, said device allowing, when contacted witha sample containing at least one nucleic acid molecule under conditionsallowing hybridisation to occur, the determination of the presence orabsence of said differentially spliced gene product.
 2. The device ofclaim 1, wherein said first and second oliponucleotide molecules areavailable from a compilation of published sequences or sequenceinformation from at least one database.
 3. The device of claim 1,wherein the support material is selected from the group consisting of afilter, a membrane and a chip.
 4. The device of claim 1, wherein saidsingle-stranded oligonucleotides are RNA or DNA molecules.
 5. The deviceof claim 1, wherein said single-stranded oligonucleotides compriseoligonucleotides of less than 50 nucleotides in length.
 6. The device ofclaim 1, wherein said single-stranded oligonucleotides are specific foralternative splicings representative of a cell or tissue in a givenpathological condition.
 7. The device of claim 6, wherein saidsingle-stranded oligonucleotides are specific for alternative splicingsrepresentative of a tumor cell or tissue.
 8. The device of claim 6,wherein said single-stranded oligonucleotides are specific foralternative splicings representative of a cell or tissue undergoingapoptosis.
 9. The device of claim 1, where said device is useful toevaluate the toxicity of a compound or treatment to a cell, tissue, ororganism by determining the presence or absence of said differentiallyspliced gene product in a sample treated with said compound ortreatment.
 10. The device of claim 1, where said device is useful toevaluate the therapeutic efficacy of a compound to a cell, tissue, ororganism by determining the presence or absence of said differentiallyspliced gene product in a sample from said cell, tissue, or organism.11. The device of claim 1, where said device is useful to evaluate theresponsiveness of a subject to a compound or treatment by determiningthe presence or absence of said differentially spliced gene product in asample from said subject exposed to said compound or treatment.
 12. Amethod of producing a device comprising a support material andsingle-stranded oligonucleotide of between 5 and 100 nucleotides inlength attached to said solid support material, wherein said methodcomprises: (a) providing said oligonucleotides, wherein saidoligonucleotides comprise at least a first and a second oligonucleotidemolecule, wherein said first oligonucleotide molecule comprises a firstsequence that is complementary to and specific for an exon or an intronof a first gene, and wherein said first sequence corresponds to a regionof variability in at least one product of said first gene due todifferential splicing, and wherein said second oligonucleotide moleculecomprises a second sequence that is complementary to and specific for anexon-exon or exon-intron junction region of said first gene, and whereinsaid second sequence corresponds to a region of variability in at leastone product of said first gene due to differential splicing; and (b)arranging and immobilizing said oligonucleotides serially on saidsupport material, said device allowing, when contacted with a samplecontaining at least one nucleic acid molecule under conditions allowinghybridisation to occur, the determination of the presence or absence ofat least one differentially spliced gene product.
 13. The method ofclaim 12, wherein said first or second oligonucleotide molecule isobtained by a method comprising: (a) identifying at least two differentoligonucleotides corresponding to a differentially spliced domain of agene, wherein said differentially spliced domain is characteristic of aphysiopathological condition, and (b) synthesizing one or severalsingle-stranded oligonucleotides complementary to and specific for saiddomain or a junction region formed by the splicing or absence ofsplicing of said domain.
 14. The method of claim 13, wherein theidentification step (a) comprises: i) hybridizing a plurality ofdifferent RNA or cDNA molecules derived from a first sample, wherein thecomposition or sequence of the RNA or cDNA molecules is at leastpartially unknown, with a plurality of different cDNA molecules derivedfrom RNA molecules of a second sample, wherein the composition orsequence of the cDNA molecules is at least partially unknown; and ii)identifying, from the hybrids formed in i), a population of nucleic acidmolecules comprising an unpaired region, wherein said unpaired regioncorresponds to a region of a gene that is differentially spliced betweensaid first and second sample.
 15. The method of claim 12, wherein saidfirst and second oligonucleotide molecules are obtained from acompilation of published sequences or sequence information fromdatabases.
 16. The method of claim 12, wherein the support material isselected from a filter, a membrane, and a chip.
 17. The method of claim12, wherein said single-stranded oligonucleotides are specific foralternative splicings representative of a cell or tissue in a givenpathological condition.
 18. The method of claim 17, wherein saidsingle-stranded oligonucleotides are specific for alternative splicingsrepresentative of a tumor cell or tissue.
 19. The method of claim 17,wherein said single-stranded oligonucleotides are specific foralternative splicings representative of a cell or tissue undergoingapoptosis.
 20. The method of claim 12, wherein said single-strandedoligonucleotides comprise oligonucleotides of less than 50 nucleotidesin length.
 21. The device of claim 1, wherein said device allows thedetermination of the presence or absence of two or more differentiallyspliced gene products of said first gene.
 22. The device of claim 1,wherein said device allows the determination of the presence or absenceof one or more differentially spliced gene products of two or moregenes.